CN108152357A - The method of liver cancer syndrome of spleen deficiency biological marker analyte detection based on sialoprotein matter group - Google Patents

The method of liver cancer syndrome of spleen deficiency biological marker analyte detection based on sialoprotein matter group Download PDF

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CN108152357A
CN108152357A CN201711241298.5A CN201711241298A CN108152357A CN 108152357 A CN108152357 A CN 108152357A CN 201711241298 A CN201711241298 A CN 201711241298A CN 108152357 A CN108152357 A CN 108152357A
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吴正治
曹美群
丁峰
孙珂焕
黄飞娟
范大华
姚永超
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Shenzhen Institute of Gerontology
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Abstract

The invention discloses the methods of the liver cancer syndrome of spleen deficiency biological marker analyte detection based on sialoprotein matter group, are as follows：Sample protein extracts；The protein concentration of extraction is measured using Brad ford methods；Proteolysis；ITRAQ is marked；Vacuum refrigeration centrifugal drying；Reversed phase chromatography separation under the conditions of high pH；ABI 5600 carries out protein analysis；MASS SPECTRAL DATA ANALYSIS：When selecting database, if biology has been sequenced, the species database is directly selected, if non-sequencing biology, is then selected and the mostly concerned major class proteome databases of sample；The mass spectral analysis of iTRAQ is carried out using 5600 type mass spectrographs of ABI.The present invention can quick, reliable proteomic techniques scheme find the specific proteins marker in liver cancer syndrome of spleen deficiency, establish a kind of check and evaluation means of noninvasive, easy, quick practicality for the early stage diagnosis and treatment of liver cancer syndrome of spleen deficiency, postoperative recurrence, transfer and Observation On The Prognosis.

Description

The method of liver cancer syndrome of spleen deficiency biological marker analyte detection based on sialoprotein matter group
Technical field
The present invention relates to a kind of biological marker object detecting method, specifically a kind of liver cancer spleen based on sialoprotein matter group The method of asthenic symptoms biological marker analyte detection.
Background technology
With the completion of the Human Genome Project (human genomic project, HGP), people will gradually study target Research to transformation to protein group (proteome).One word of protein group is to be carried in 1994 by Williams and Wilkins Go out, refer to all proteins of genome encoding, i.e., a certain species, individual, organ, tissue or even cell all protein, by force The entirety that the corresponding all proteins of genome is adjusted to form.2001, Nature and Science were announcing human genome grass While figure, commentary and the prospect of Abbott and Fields have been delivered respectively.Therewith, proteomics has come up Unprecedented height, it is considered to be the commanding elevation of functional genomics forward position research strategy, decisive point.Proteomics (proteomics) it is a kind of emerging scientific research technology, is different from the diagnostic cast of traditional individual gene or single albumen Formula, but analyze body or cell all protein constituent, expression, modification, structure function and phase interaction from integral level With and interaction between protein and nucleic acid, have comprehensive and essential understanding to the complicated activity of life, from entirety Dynamic studies are carried out to protein on level.
Protein is the executor of vital movement, is the product of gene expression, expression, modification, function and phase each other Interaction by heredity and environmental factor influenced, then body occur morbid state will necessarily cause protein content or The variation of person's existence form, the protein molecule of these variations can provide foundation for the material base of clinical disease diagnosis.Egg White matter group is mainly with modern advanced detection technique, and detection body is from small to one cell to greatly to entire body Difference under the characteristic of protein expressed by full gene, comparative analysis physiology or pathological state, medication and not medication state Protein is expressed, finds out the protein of the next body differential expression of different conditions, thus the morning of the material base, disease for life Phase is detected finds molecular diagnostic markers offer foundation with researchs such as diagnosis.
In recent years, with the development innovation of modern science and biochemical trace detection analytical technology, saliva is as one Kind clinical easily acquisition and operating process have progressed into the visual angle of people's research activities without traumatic body fluid completely, And with inestimable Scientific Research Potential and potential applicability in clinical practice.First, for compared to serum specimen, saliva sample is adopted Collect safer, have it is non-invasive, patient's no pain in gatherer process is easy to receive, and without blood borne disease propagate risk In the presence of；Compared with urine specimen, saliva sample has the advantages that can real-time sampling, more easily.In addition, what saliva detection needed Sample size is small, at low cost, easily stored and transport.Most of all, the body fluid components such as saliva sample ingredient and blood, urine With high similitude, there is extremely strong sensibility and specificity to medical diagnosis on disease.
Saliva is a kind of common important body fluid of human body, forgives a large amount of hormone, protein, enzyme, antibody, complement, thin The blood constituents such as intracellular cytokine and various microorganisms, these blood constituents by along concentration gradient it is passive cross-cell membrane transhipment or it is inverse The approach such as the active transport of concentration gradient are secreted in saliva, and are in dynamic change with the influence of internal pathophysiological change Process.Study the composition transfers such as pH value, electrolyte, biochemical indicator, microorganism, immune indexes, protein and disease of saliva Relationship, find salivary component variation can be used as medical diagnosis on disease, curative effect judgement, drug surveillance reference index, in clinical disease Diagnosis and treatment activity in have important potential using value.It is shown according to current documents and materials, saliva can not only be used as Chinese mugwort Grow the diagnostic markers of the malignant tumours such as communicable diseases, malignant tumor of mouth and the breast cancer such as disease, hepatitis B, Er Qieke To be applied to the diagnosis of each systemic disease such as diabetes, arthritis, heart disease and kidney trouble, so as to as current west The preferred sample of substitution Serological testing that national researcher is keen to.
Saliva has the biological works such as digestion food, lubrication, defence protection, buffering, mechanical cleaning, antibacterial and endocrine With, and proteins and peptides are the most important substances with biological function in salivary component, have now been found that sialoprotein Matter and polypeptide have more than 2300 kinds, mainly have alpha-amylase, albumin, cysteine proteinase, IgA, lysozyme, lactoferrin, The protein such as mucin, wherein the sialoprotein matter for having 98% is Statherin and transferrins.However, by pair it has been found that The function of sialoprotein matter classify, the unknown protein of discovery feature occupies 28.7% (ratio is maximum), with immune work( The relevant protein of energy accounts for 21%, replicates with protein and repair relevant protein accounting for 1.6%, with cell mobility and secretion Relevant protein accounts for 4.8%, 2.3% is accounted for transcription and the relevant protein of ribosomes, with cell proliferation and cell cycle phase What is closed accounts for 4.2%, and account for 9.7% respectively with the relevant protein of signal transduction, metabolism, cytoskeleton and inner membrance, 5.2%, 7.1% etc..As it can be seen that the biological function still sialoprotein matter in unknown state still occupies sizable ratio, need further Research is excavated, and significant.Recently research confirm, big flux, big precision proteomic techniques application, make saliva egg White matter biomarker becomes for the early diagnosis prevention of disease, bioprotein targeted therapy, Prognosis scoveillance judgement etc. can Energy.
Invention content
The purpose of the present invention is to provide a kind of safe and reliable, high efficiency liver cancer spleens based on sialoprotein matter group The method of asthenic symptoms biological marker analyte detection, to solve the problems mentioned in the above background technology.
To achieve the above object, the present invention provides following technical solution：
The method of liver cancer syndrome of spleen deficiency biological marker analyte detection based on sialoprotein matter group, is as follows：
(1) sample protein extracts：The lysis buffer that 5% is added in sample carries out vortex mixing, ultrasonic 60s, amplitude 22% room temperature extracts 30min；15000r/min, 4 DEG C of centrifugation 20min, take out supernatant；Collect supernatant, packing packing after freeze in -80℃；
(2) protein quantification：The protein concentration of extraction is measured using Bradford methods, first mixes the sample with lysis buffer progress Certain multiple dilution its final concentration is made to fall in the range of mark song, the sample and standard items diluted respectively take 10 μ l respectively with 300 μ l Protein quantification dyestuff is protected from light 20min, with microplate reader simultaneously the light absorption value of bioassay standard product and sample under 595nm, according to Often the relationship of pipe light absorption value and concentration draws standard curve to standard items, and each sample protein concentration is calculated according to curve equation；
(3) proteolysis：200 μ g protein solutions is taken to be placed in centrifuge tube after protein quantification；Add in 4 μ l reducing agents, 60 DEG C React 1h；The cysteine blocking agent of 2 μ l is added in, reacts at room temperature 10min；Protein solution after reductive alkylation is added in into 10K Super filter tube in, 12000r/min centrifugation 20min, discard collecting pipe bottom solution；The solution added in iTRAQ kits delays 100 μ l of fliud flushing, 12000 leave heart 20min, discard collecting pipe bottom solution, are repeated 3 times；The collecting pipe more renewed, in ultrafiltration Trypsase is added in pipe, 4 μ g of total amount, 50 μ l of volume, 37 DEG C of reactions are overnight；Next day, 12000r/min centrifugation 20min, enzymolysis Postdigestive peptide fragment solution centrifugation is in collection bottom of the tube；50 μ l dissolving buffer solutions are added in super filter tube, 12000r/min is again 20min is centrifuged, is merged with upper step, collects the sample that bottom of the tube is obtained after 100 μ l enzymolysis；
(4) iTRAQ is marked：ITRAQ reagents are taken out from refrigerator, room temperature is equilibrated to, iTRAQ reagents is centrifuged to tube bottom； 150 μ l isopropanols are added in into every pipe iTRAQ reagents, vortex oscillation is centrifuged to tube bottom；Take the sample transfer after 50 μ l enzymolysis Into new centrifuge tube；ITRAQ reagents are added in sample, vortex oscillation, centrifuge to tube bottom, react at room temperature 2h；It adds in 100 μ l water terminate reaction；1ul mixing is respectively taken out from 4 groups of samples, with carrying out MALDI-TOF-TOF mirror after Ziptip desalinations Fixed, confirmation flag reaction is good；Sample after mixed mark, vortex oscillation are centrifuged to tube bottom；Vacuum refrigeration centrifugal drying；It takes out Sample freezen protective after dry is for use；
(5) reversed phase chromatography separation under the conditions of high pH：The allotment of required reagent：Mobile phase A：98%ddH2O, 2% second Nitrile, pH10；Mobile phase B：98% acetonitrile, 2%ddH2O, pH10；DdH2O ammonium hydroxide tune pH value to 10；Sample after mixed mark Product 100ul mobile phase As dissolve, and 14000r/min centrifugation 20min take supernatant for use；It is carried out using the BSA that 400 μ g have been digested Separation, detecting system situation；Take the ready sample loadings of 100ul；Flow velocity 0.7ml/min obtains separation gradient data；
(6) ABI-5600 carries out protein analysis：The allotment of required reagent：Mobile phase A：100% ultra-pure water, 0.1% first Acid；Mobile phase B：100% acetonitrile, 0.1% formic acid；By the component that high pH reverse phase separations obtain 20 μ l2% methanol, 0.1% first Acid redissolves；12000r/min centrifuges 10min, draws supernatant loading, and 10 μ l of loading volume take sandwich method loading；Loading pump stream Fast 350nl/min, 15min；Flow velocity 350nl/min is detached, obtains separation gradient data；
(7) MASS SPECTRAL DATA ANALYSIS：When selecting database, if biology has been sequenced, the species database is directly selected, If non-sequencing biology, then select and the mostly concerned major class proteome databases of sample；Using ABI-5600 type matter Spectrometer carries out the mass spectral analysis of iTRAQ；
(8) testing result：Contrast experiment, quantitative proteomics research knot are carried out to liver cancer syndrome of spleen deficiency and Normal group Fruit shows：Identify 1297 albumen；Compared with Normal group, 1.5 times or more differential expressions being identified in liver cancer syndrome of spleen deficiency Totally 140, significant difference albumen, wherein upregulated protein 64, down-regulation protein 76.Differential protein is listed as follows：Raise egg It is white as follows：
1) searching number is Q66K66, and gene annotation is Transmembrane protein 198；
2) searching number is Q9ULD9, and gene annotation is Zinc finger protein 608；
3) searching number is Q8IVF7, and gene annotation is Formin-like protein 3；
4) searching number is O60282, and gene annotation is Kinesin heavy chain isoform 5C；
5) searching number is Q86V2, and gene annotation is Kinesin-like protein KIF27；
6) searching number is P06727, and gene annotation is Apolipoprotein A-IV；
7) searching number is P13473, and gene annotation is Lysosome-associated membrane glycoprotein 2；
8) searching number is P31151, and gene annotation is Protein S100-A7；
9) searching number is O75629, and gene annotation is Protein CREG1；
10) searching number is P35321, gene annotation Cornifin-A；
11) searching number is Q9Y239, and gene annotation is Nucleotide-binding oligomerization domain-containing protein 1；
12) searching number is Q9BRA2, and gene annotation is Thioredoxin domain-containing protein 17；
13) searching number is Q07654, and gene annotation is Trefoil factor 3；
14) searching number is P31949, and gene annotation is Protein S100-A11；
15) searching number is Q96RM1, and gene annotation is Small proline-rich protein 2F；
16) searching number is Q6ZUA9, and gene annotation is Maestro heat-like repeat family member 5；
17) searching number is P68032, gene annotation Actin, alpha cardiac musle 1；
18) searching number is P01598, and gene annotation is Ig kappa chain V-I region EU；
19) searching number is P01708, and gene annotation is Ig lambda chain V-II region BUR；
20) searching number is P83593, and gene annotation is Ig kappa chain V-IV region STH (Fragment)；
21) searching number is P02814, and gene annotation is Submaxillary gland androgen-regulated protein 3B；
22) searching number is Q6P158, and gene annotation is Putative ATP-dependent RNA helicase DH 57；
23) searching number is Q9UBH0, and gene annotation is Interleukin-36receptor antagonist protein；
24) searching number is P49221, and gene annotation is Protein-glutamine gamma- glutamyltransferase 4；
25) searching number is P00480, and gene annotation is Ornithine carbamoyltransferase, mitochondrial；
26) searching number is P68871, and gene annotation is Hemoglobin subunit beta；
27) searching number is P01871, and gene annotation is Ig mu chain C region；
28) searching number is O75038, and gene annotation is 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase eta-2；
29) searching number is P31025, gene annotation Lipocalin-1；
30) searching number is P01612, and gene annotation is Ig kappa chain V-I region Mev；
31) searching number is P19957, gene annotation Elafin；
32) searching number is P08697, gene annotation Alpha-2-antiplasmin；
33) searching number is P32119, gene annotation Peroxiredoxin-2；
34) searching number is Q7L0X2, and gene annotation is Glutamate-rich protein 6；
35) searching number is Q9UL2, and gene annotation is Ras-related protein Rab-21；
36) searching number is P22528, gene annotation Cornifin-B；
37) searching number is Q9Y5Y6, and gene annotation is Suppressor of tumorigenicity 14protein；
38) searching number is P69905, and gene annotation is Hemoglobin subunit alpha；
39) searching number is P01743, and gene annotation is；Ig heavy chain V-I region HG3；
40) searching number is Q6ZP82, and gene annotation is Coiled-coil domain-containing protein 141；
41) searching number is Q01469, and gene annotation is Fatty acid-binding protein, epidermal；
42) searching number is P01860, and gene annotation is Ig gamma-3chain C region；
43) searching number is P09211, and gene annotation is Glutathione S-transferase P；
44) searching number is Q8TED4, and gene annotation is Sugar phosphate exchanger 2；
45) searching number is P04179, and gene annotation is Superoxide dismutase [Mn], mitochondrial；
46) searching number is P01624, and gene annotation is Ig kappa chain V-III region POM；
47) searching number is P1059, gene annotation Thioredoxin；
48) searching number is O95274, and gene annotation is Ly6/PLAUR domain-containing protein 3；
49) searching number is Q9GZN4, and gene annotation is Brain-specific serine protease 4；
50) searching number is Q9ULV4, gene annotation Coronin-1C；
51) searching number is P35270, and gene annotation is Sepiapterin reductase；
52) searching number is Q86SG5, and gene annotation is Protein S100-A7A；
53) searching number is P01764, and gene annotation is Ig heavy chain V-III region 23；
54) searching number is P06870, gene annotation Kallikein-1；
55) searching number is Q96PD5, and gene annotation is N-acetylmuramoyl-L-alanine amidase；
56) searching number is Q9UHY7, and gene annotation is Enolase-phosphatase E1；
57) searching number is Q3ZCW2, and gene annotation is Galectin-related protein；
60) searching number is Q92692, gene annotation Nectin-2；
61) searching number is P01834, and gene annotation is Ig kappa chain C region；
62) searching number is P00915, and gene annotation is Carbonic anhydrase 1；
63) searching number is P01771, and gene annotation is Ig heavy chain V-III region HIL；
64) searching number is P01857, and gene annotation is Ig gamma-1chain C region；
65) searching number is P04220, and gene annotation is Ig mu heavy chain disease protein；
66) searching number is P00450, gene annotation Ceruloplasmin；
67) searching number is P00734, gene annotation Prothrombin；
68) searching number is Q6MZM9, and gene annotation is Proline-richprotein 27；
69) searching number is P01610, and gene annotation is Ig kappa chain V-I region WEA；
70) searching number is P02763, and gene annotation is Alpha-1-acid glycoprotein 1；
71) searching number is P15328, and gene annotation is Folate receptor alpha；
72) searching number is P02042, and gene annotation is Hemoglobin subunit delta；
73) searching number is O60701, and gene annotation is UDP-glucose 6-dehydrogenase；
74) searching number is O00154, and gene annotation is Cytosolic acylcoenzyme A thioester hydrolase；
75) searching number is P41439, and gene annotation is Folate receptor gamma；
76) searching number is Q14126, gene annotation Desmoglein-2；
77) searching number is Q92734, gene annotation ProteinTFG；
78) searching number is Q92876, gene annotation Kallikrein-6；
79) searching number is O60635, gene annotation Tetraspanin-1；
80) searching number is P19404, and gene annotation is NADHdehydrogenase [ubiquinone] flavoprotein 2, mitochondrial；
81) searching number is P02787, gene annotation Serotransferrin；
82) searching number is P22532, and gene annotation is Smallproline-rich protein 2D；
83) searching number is B3EWG3, and gene annotation is Protein FAM25A；
84) searching number is P68402, and gene annotation is Platelet-activating factor acetylhydrolase IB subunit beta；
85) searching number is O75506, and gene annotation is Heat shockfactor-binding protein 1；
86) searching number is P01770, and gene annotation is Ig heavy chain V-III region NIE；
87) searching number is Q8IVW1, and gene annotation is ADP-ribosylation factor-like protein 17；
88) searching number is P01040, gene annotation Cystatin-A；
89) searching number is P04217, gene annotation Alpha-1B-glycoprotein；
90) searching number is P04181, and gene annotation is Ornithine aminotransferase, mitochondrial；
91) searching number is P05388, and gene annotation is 60S acidic ribosomal protein P0；
92) searching number is Q15223, gene annotation Nectin-1；
93) searching number is P0C870, and gene annotation is JmjC domain-containing protein 7；
94) searching number is P20160, gene annotation Azurocidin；
95) searching number is P02768, and gene annotation is Serum albumin；
96) searching number is O15231, and gene annotation is Zinc finger protein 185；
97) searching number is Q96PF2, and gene annotation is Testis-specific serine/threonine-protein kinase 2；
98) searching number is Q8NI51, and gene annotation is Transcriptional repressor CTCFL；
99) searching number is P01859, and gene annotation is Ig gamma-2chain C region；
100) searching number is P02760, and gene annotation is Protein AMBP；
101) searching number is P08236, gene annotation Beta-glucuronidase；
102) searching number is Q9Y3D6, and gene annotation is Mitochondrial fission 1protein；
103) searching number is P80188, and gene annotation is Neutrophil gelatinase-associated lipocalin；
104) searching number is Q6UW32, and gene annotation is Insulin growth factor-like family member 1；
105) searching number is O75131, gene annotation Copine-3；
106) searching number is Q04760, and gene annotation is Lactoylglutathione lyase；
107) searching number is Q9BPV8, and gene annotation is P2Y purinoceptor 13；
108) searching number is P58062, and gene annotation is Serine protease inhibitor Kazal-type 7；
109) searching number is P02749, and gene annotation is Beta-2-glycoprotein 1；
110) searching number is P02774, and gene annotation is Vitamin D-binding protein；
111) searching number is P01762, and gene annotation is Ig heavy chain V-III region TRO；
112) searching number is P00738, gene annotation Haptoglobin；
113) searching number is P01767, and gene annotation is Ig heavy chain V-III region BUT；
114) searching number is P02671, and gene annotation is Fibrinogen alpha chain；
115) searching number is P04003, and gene annotation is C4b-binding protein alpha chain；
116) searching number is Q96L46, and gene annotation is Calpain small subunit 2；
117) searching number is Q9NZH8, gene annotation Interleukin-36gamma；
118) searching number is P31944, gene annotation Caspase-14；
119) searching number is P02753, and gene annotation is Retinol-binding protein 4；
120) searching number is Q13740, gene annotation CD166antigen；
121) searching number is Q8WWY7, and gene annotation is WAP four-disulfide core domain protein 12；
122) searching number is O75116, and gene annotation is Rho-associated protein kinase 2；
123) searching number is Q14210, and gene annotation is Lymphocyte antigen 6D；
124) searching number is P0CG39, and gene annotation is POTE ankyrin domain family member J；
125) searching number is Q9UKR3, gene annotation Kallikrein-13；
126) searching number is P01605, and gene annotation is Ig kappa chain V-I region Lay；
127) searching number is P01778, and gene annotation is Ig heavy chain V-III region ZAP；
128) searching number is P51159, and gene annotation is Ras-related protein Rab-27A；
129) searching number is P02790, gene annotation Hemopexin；
130) searching number is P01781, and gene annotation is Ig heavy chain V-III region GAL；
131) searching number is P17516, and gene annotation is Aldo-keto reductase family 1member C4；
132) searching number is P06731, and gene annotation is Carcinoembryonic antigen-related cell adhesion molecule 5；
133) searching number is P05155, and gene annotation is Plasma protease C1inhibitor；
134) searching number is P28799, gene annotation Granulins；
135) searching number is Q02487, gene annotation Desmocollin-2；
136) searching number is Q96AT9, and gene annotation is Ribulose-phosphate 3-epimerase；
137) searching number is O00748, and gene annotation is Cocaine esterase；
138) searching number is P29508, and gene annotation is Serpin B3；
139) searching number is Q14CN2, and gene annotation is Calcium-activated chloride channel regulator 4；
140) searching number is P0C0L5, and gene annotation is Complement C4-B；
141) searching number is P01776, and gene annotation is Ig heavy chain V-III region WAS；
142) searching number is P12830, gene annotation Cadherin-1；
143) searching number is Q14574, gene annotation Desmocollin-3；
144) searching number is P01623, and gene annotation is Ig kappa chain V-III region WOL；
145) searching number is Q8N271, gene annotation Prominin-2；
146) searching number is P01601, and gene annotation is Ig kappa chain V-ID region 16 (Fragment)；
147) searching number is Q12792, gene annotation Twinfilin-1；
148) searching number is P69891, and gene annotation is Hemoglobin subunit gamma-1；
149) searching number is P30048, and gene annotation is Thioredoxin-dependent peroxide reductase,mitochondrial；
150) searching number is P08603, and gene annotation is Complement factor H；
151) searching number is P02788, gene annotation Lactotransferrin；
152) searching number is O75347, and gene annotation is Tubulin-specific chaperone A；
153) searching number is P01023, gene annotation Alpha-2-macroglobulin；
154) searching number is P06748, gene annotation Nucleophosmin；
155) searching number is P01833, and gene annotation is Polymeric immunoglobulin receptor；
156) searching number is P04632, and gene annotation is Calpain small subunit 1；
157) searching number is P24158, gene annotation Myeloblastin；
158) searching number is P19971, and gene annotation is Thymidine phosphorylase；
Down-regulation protein is as follows：
1) searching number is Q12955, gene annotation Ankyrin-3；
2) searching number is O95716, and gene annotation is Ras-related protein Rab-3D；
3) searching number is Q2KHT4, and gene annotation is Germ cell-specific gene 1protein；
4) searching number is Q13043, and gene annotation is Serine/threonine-protein kinase 4；
5) searching number is F5H4A9, and gene annotation is Uncharacterized membrane protein C3orf80；
6) searching number is Q8IWB9, and gene annotation is Testis-expressed sequence 2protein；
7) searching number is P01602, and gene annotation is Ig heavy chain V-I region 5 (Fragment)；
8) searching number is Q8TC84, and gene annotation is Fibronectin type 3and ankyrinrepeat domains protein 1；
9) searching number is Q8IV20, and gene annotation is Laccase domain-containing protein 1；
10) searching number is P08754, and gene annotation is Guanine nucleotide-binding protein G (k) subunit alpha；
11) searching number is Q6T4P5, and gene annotation is Lipid phosphate phosphatase-related proteintype 3；
12) searching number is P50748, and gene annotation is Kinetochore-associated protein 1；
13) searching number is Q8IVL1, and gene annotation is Neuronnavigator 2；
14) searching number is P60953, and gene annotation is Cell division control protein 42homolog；
15) searching number is P02808, gene annotation Statherin；
16) searching number is Q9UKK3, and gene annotation is Poly [ADP-ribose] polymerase 4；
17) searching number is P78371, and gene annotation is T-complex protein 1subunit beta；
18) searching number is O00602, gene annotation Ficolin-1；
19) searching number is P22392, and gene annotation is Nucleoside diphosphate kinase B；
20) searching number is Q9BRZ2, and gene annotation is E3ubiquitin-protein ligase TRIM56；
21) searching number is O75015, and gene annotation is Low affinity immunoglobulin gamma Fc region receptor III-B；
22) searching number is Q15493, gene annotation Regucalcin；
23) searching number is P01703, and gene annotation is Ig lambda chain V-I region NEWM；
24) searching number is P16401, and gene annotation is Histone H1.5；
25) searching number is P80303, gene annotation Nucleobindin-2；
26) searching number is P32243, and gene annotation is Homeobox protein OTX2；
27) searching number is Q16378, and gene annotation is Proline-rich protein 4；
28) searching number is Q7Z5M8, and gene annotation is Abhydrolase domain-containing protein 12B；
29) searching number is P58499, and gene annotation is Protein FAM3B；
30) searching number is P42765, and gene annotation is 3-ketoacyl-CoA thiolase, mitochondrial；
31) searching number is Q9P0M2, and gene annotation is A-kinase anchor protein 7isoform gamma；
32) searching number is P22626, and gene annotation is Heterogeneous nuclear ribonucleoproteins A2/B1；
33) searching number is Q9UNU6, and gene annotation is 7-alpha-hydroxycholest-4-en-3-one 12- alpha-hydroxylase；
34) searching number is P20929, gene annotation Nebulin；
35) searching number is P04259, gene annotation Keratin, type II cytoskeletal 6B；
36) searching number is P04438, and gene annotation is Ig heavy chain V-II region SESS；
37) searching number is O43852, gene annotation Calumenin；
38) searching number is Q9UHY1, and gene annotation is Nuclear receptor-binding protein；
39) searching number is Q12841, and gene annotation is Follistatin-related protein 1；
40) searching number is Q9UPT6, and gene annotation is C-Jun-amino-terminal kinase-interacting protein 3；
41) searching number is Q06828, gene annotation Fibromodulin；
42) searching number is Q9H4M9, and gene annotation is EH domain-containing protein 1；
43) searching number is Q04917 14-3-3, and gene annotation is protein eta；
44) searching number is Q9HCU9, and gene annotation is Breast cancer metastasis-suppressor 1；
45) searching number is P05089, gene annotation Arginase-1；
46) searching number is Q96RS0, and gene annotation is Trimethylguanosine synthase；
47) searching number is Q8IWF6, and gene annotation is Protein DENND6A；
48) searching number is Q92521, and gene annotation is GPI mannosyltransferase 3；
49) searching number is P33241, and gene annotation is Lymphocyte-specific protein 1；
50) searching number is Q96FZ7, and gene annotation is Charged multivesicular body protein 6；
51) searching number is Q9ULZ3, and gene annotation is Apoptosis-associated speck-like protein containing a CARD；
52) searching number is P25787, and gene annotation is Proteasome subunit alpha type-2；
53) searching number is O60361, and gene annotation is Putative nucleoside diphosphate kinase；
54) searching number is Q8TAX7, gene annotation Mucin-7；
55) searching number is Q92688, and gene annotation is Acidic leucine-rich nuclear phosphoprotein 32 family member B；
56) searching number is P40394, and gene annotation is Alcohol dehydrogenase class 4mu/sigma chain；
57) searching number is Q14289, and gene annotation is Protein-tyrosine kinase 2-beta；
58) searching number is P55008, and gene annotation is Allograft inflammatory factor 1；
59) searching number is P08571, and gene annotation is Monocyte differentiation antigen CD14；
60) searching number is P21439, and gene annotation is Multidrug resistance protein 3；
61) searching number is O95777U6, and gene annotation is snRNA-associated Sm-like protein LSm8；
62) searching number is Q96DA0, and gene annotation is Zymogen granule protein 16homolog B；
63) searching number is Q16651, gene annotation Prostasin；
64) searching number is Q9Y4Y9, and gene annotation is U6snRNA-associated Sm-like protein LSm5；
65) searching number is P26583, and gene annotation is High mobility group protein B2；
66) searching number is Q9H008, and gene annotation is Phospholysine phosphohistidine inorganic pyrophosphate phosphatase；
67) searching number is P01742, and gene annotation is Ig heavy chain V-I region EU；
68) searching number is Q15631, gene annotation Translin；
69) searching number is Q9HC38, and gene annotation is Glyoxalase domain-containing protein 4；
70) searching number is Q9Y376, and gene annotation is Calcium-binding protein 39；
71) searching number is Q96DR5, and gene annotation is BPI fold-containing family A member 2；
72) searching number is P07477Trypsin-1；
73) searching number is Q96C19, and gene annotation is EF-hand domain-containing protein D2；
74) searching number is P16870, and gene annotation is Carboxypeptidase E；
75) searching number is Q8NI35, and gene annotation is InaD-like protein；
76) searching number is P05362, and gene annotation is Intercellular adhesionmolecule 1；
77) searching number is P15374, and gene annotation is Ubiquitin carboxyl-terminalhydrolase isozyme L3；
78) searching number is Q6ZNW5, and gene annotation is GDP-D-glucose phosphorylase 1；
79) searching number is Q15370, and gene annotation is Transcriptionelongationfactor B polypeptide 2；
80) searching number is P0CG05, and gene annotation is Iglambda-2chain C regions；
81) searching number is P30740, and gene annotation is Leukocyte elastase inhibitor；
82) searching number is Q7Z6J4, gene annotation FYVE, RhoGEF and PH domain-containing protein 2；
83) searching number is Q9H7L9, and gene annotation is Sin3histone deacetylase corepressor complex component SDS3；
84) searching number is P00533, and gene annotation is Epidermal growthfactor receptor；
85) searching number is P30046, and gene annotation is D-dopachrome decarboxylase；
86) searching number is Q96BQ1, and gene annotation is Protein FAM3D；
87) searching number is Q9NP98, gene annotation Myozenin-1；
88) searching number is P31146, gene annotation Coronin-1A；
89) searching number is O75503, and gene annotation is Ceroid-lipofuscinosis neuronal protein 5；
90) searching number is P63279, and gene annotation is SUMO-conjugating enzyme UBC9；
91) searching number is Q8IUE6, and gene annotation is Histone H2A type 2-B；
92) searching number is Q9NZ53, and gene annotation is Podocalyxin-like protein 2；
93) searching number is Q8NF91, gene annotation Nesprin-1；
94) searching number is Q9BS40, gene annotation Latexin；
95) searching number is P09382, gene annotation Galectin-1；
96) searching number is Q9BRF8, and gene annotation is Serine/threonine-protein phosphatase CPPED1；
97) searching number is P08579, and gene annotation is U2small nuclear ribonucleoprotein B "；
98) searching number is P49720, and gene annotation is Proteasome subunit beta type-3；
99) searching number is P52566, and gene annotation is Rho GDP-dissociation inhibitor 2；
100) searching number is Q8NC60, and gene annotation is Nitric oxide-associated protein 1；
101) searching number is P10909, gene annotation Clusterin；
102) searching number is P01712, and gene annotation is g lambda chain V-II region WIN；
103) searching number is P23280, and gene annotation is Carbonic anhydrase 6；
104) searching number is Q8IWQ3, and gene annotation is Serine/threonine-protein kinase BRSK2；
105) searching number is Q86VR7, and gene annotation is V-set and immunoglobulin domain- containing protein 10-like；
106) searching number is Q99439, gene annotation Calponin-2；
107) searching number is Q9GZZ8, and gene annotation is Extracellular glycoprotein lacritin；
108) searching number is O43707, gene annotation Alpha-actinin-4；
109) searching number is P62253, and gene annotation is Ubiquitin-conjugating enzyme E2G1；
110) searching number is Q13421, gene annotation Mesothelin；
111) searching number is P35237, and gene annotation is Serpin B6；
112) searching number is P14550, and gene annotation is Alcohol dehydrogenase [NADP (+)]；
113) searching number is P06737, and gene annotation is Glycogen phosphorylase, liver form；
114) searching number is O00584, and gene annotation is Ribonuclease T2；
115) searching number is P62993, and gene annotation is Growth factor receptor-bound protein2；
116) searching number is P01707, and gene annotation is Ig lambda chain V-II region TRO；
117) searching number is P01036, gene annotation Cystatin-S；
118) searching number is P35908, gene annotation Keratin, type II cytoskeletal 2epidermal；
119) searching number is O95684, and gene annotation is FGFR1oncogene partner；
120) searching number is O75891, and gene annotation is Cytosolic 10-formyltetrahydrofolate dehydrogenase；
121) searching number is Q86VD1, and gene annotation is MORC family CW-type zinc finger protein 1；
122) searching number is Q9Y490, gene annotation Talin-1；
123) searching number is P05154, and gene annotation is Plasma serine protease inhibitor；
124) searching number is O15143, and gene annotation is Actin-related protein 2/3complex subunit 1B；
125) searching number is O75563, and gene annotation is Src kinase-associated phosphoprotein 2；
126) searching number is A0M8Q6, and gene annotation is Ig lambda-7chain C region；
127) searching number is Q6JBY9, and gene annotation is CapZ-interacting protein.
Compared with prior art, the beneficial effects of the invention are as follows：
The present invention can quick, reliable proteomic techniques scheme find the specific proteins in liver cancer syndrome of spleen deficiency Marker is that the early stage diagnosis and treatment of liver cancer syndrome of spleen deficiency, postoperative recurrence, transfer and Observation On The Prognosis establish a kind of noninvasive, easy, quick reality Check and evaluation means.
Description of the drawings
Fig. 1 is the basic procedure schematic diagram that iTRAQ quantitative proteomics are tested in the present invention.
Specific embodiment
The technical solution of this patent is described in more detail With reference to embodiment.
Referring to Fig. 1, the method for the liver cancer syndrome of spleen deficiency biological marker analyte detection based on sialoprotein matter group, specific steps It is as follows：
(1) sample protein extracts：The lysis buffer that 5% is added in sample carries out vortex mixing, ultrasonic 60s, amplitude 22% room temperature extracts 30min；15000r/min, 4 DEG C of centrifugation 20min, take out supernatant；Collect supernatant, packing packing after freeze in -80℃；
(2) protein quantification：The protein concentration of extraction is measured using Bradford methods, first mixes the sample with lysis buffer progress Certain multiple dilution its final concentration is made to fall in the range of mark song, the sample and standard items diluted respectively take 10 μ l respectively with 300 μ l Protein quantification dyestuff is protected from light 20min, with microplate reader simultaneously the light absorption value of bioassay standard product and sample under 595nm, according to Often the relationship of pipe light absorption value and concentration draws standard curve to standard items, and each sample protein concentration is calculated according to curve equation；
(3) proteolysis：200 μ g protein solutions is taken to be placed in centrifuge tube after protein quantification；Add in 4 μ l reducing agents, 60 DEG C React 1h；The cysteine blocking agent of 2 μ l is added in, reacts at room temperature 10min；Protein solution after reductive alkylation is added in into 10K Super filter tube in, 12000r/min centrifugation 20min, discard collecting pipe bottom solution；The solution added in iTRAQ kits delays 100 μ l of fliud flushing, 12000 leave heart 20min, discard collecting pipe bottom solution, are repeated 3 times；The collecting pipe more renewed, in ultrafiltration Trypsase is added in pipe, 4 μ g of total amount, 50 μ l of volume, 37 DEG C of reactions are overnight；Next day, 12000r/min centrifugation 20min, enzymolysis Postdigestive peptide fragment solution centrifugation is in collection bottom of the tube；50 μ l dissolving buffer solutions are added in super filter tube, 12000r/min is again 20min is centrifuged, is merged with upper step, collects the sample that bottom of the tube is obtained after 100 μ l enzymolysis；
(4) iTRAQ is marked：ITRAQ reagents are taken out from refrigerator, room temperature is equilibrated to, iTRAQ reagents is centrifuged to tube bottom； 150 μ l isopropanols are added in into every pipe iTRAQ reagents, vortex oscillation is centrifuged to tube bottom；Take the sample transfer after 50 μ l enzymolysis Into new centrifuge tube；ITRAQ reagents are added in sample, vortex oscillation, centrifuge to tube bottom, react at room temperature 2h；It adds in 100 μ l water terminate reaction；1ul mixing is respectively taken out from 4 groups of samples, with carrying out MALDI-TOF-TOF mirror after Ziptip desalinations Fixed, confirmation flag reaction is good；Sample after mixed mark, vortex oscillation are centrifuged to tube bottom；Vacuum refrigeration centrifugal drying；It takes out Sample freezen protective after dry is for use.
(5) reversed phase chromatography separation under the conditions of high pH：The allotment of required reagent：Mobile phase A：98%ddH2O, 2% second Nitrile, pH10；Mobile phase B：98% acetonitrile, 2%ddH2O, pH10；DdH2O ammonium hydroxide tune pH value to 10；Sample after mixed mark Product 100ul mobile phase As dissolve, and 14000r/min centrifugation 20min take supernatant for use；It is carried out using the BSA that 400 μ g have been digested Separation, detecting system situation；Take the ready sample loadings of 100ul；Flow velocity 0.7ml/min obtains separation gradient data；
(6) ABI-5600 carries out protein analysis：The allotment of required reagent：Mobile phase A：100% ultra-pure water, 0.1% first Acid；Mobile phase B：100% acetonitrile, 0.1% formic acid；By the component that high pH reverse phase separations obtain 20 μ l2% methanol, 0.1% first Acid redissolves；12000r/min centrifuges 10min, draws supernatant loading, and 10 μ l of loading volume take sandwich method loading；Loading pump stream Fast 350nl/min, 15min；Flow velocity 350nl/min is detached, obtains separation gradient data；
(7) MASS SPECTRAL DATA ANALYSIS：When selecting database, if biology has been sequenced, the species database is directly selected, If non-sequencing biology, then select and the mostly concerned major class proteome databases of sample；Using ABI-5600 type matter Spectrometer carries out the mass spectral analysis of iTRAQ.
ITRAQ technologies are that a kind of isotope labelling is opposite and absolute quantitation (isobaric tags for relative And absolute quantitation, iTRAQ) technology, the opposite and absolute quantitation research available for protein.This research It is based on iTRAQ technologies, joint tandem mass spectrum proteomics method ((liquid chromatography coupled With tandem mass spectrometry, LC-MS/MS), main purpose is by liver cancer syndrome of spleen deficiency and normal healthy people Group's control quantitatively detects the differential protein expression situation in saliva, while related to liver cancer syndrome of spleen deficiency syndrome for further screening Protein or protein group provide research reference, from protein level research liver cancer syndrome of spleen deficiency syndrome essence.
1. materials and methods
1.1 case selection
1.1.1 diagnostic criteria
(1) Disease Diagnosis Standard
The diagnosis of liver cancer according to《Clinical practice》Diagnostic criteria.
(2) TCM Syndrome Type diagnostic criteria
Spleen feebleness diagnosis standard：
1.1.2 1. inclusion criteria meets diagnostic criteria, and the age is between 20-75 Sui；2. non-underwent operative and chemicotherapy；3. from It is willing to and can coordinate the study subject of participation.More than 3 must all meet and can just be included in.
1.1.3 1. exclusion criteria does not meet above-mentioned diagnostic criteria person；2. 75 years old person of age ＜ 20 or ＞；3. with oral cavity office The inflammation of portion and salivary gland, digestive tract ulcer, tumour and congenital disorders；With Sjogren syndrome, cystic fibrosis；Suffer from There is other systems severe concurrent disease.
1.1.4 quality control is 1. using unified diagnostic criteria, evaluation forms lattice, unified investigation method；2. Check person is adjusted to exist It is performed when adjusting Check in strict accordance with " standardization ", reduces investigator bias, it is ensured that the consistency and authenticity of data；3. pathology knot Fruit is diagnosed by Grade III Class A hospital in January.
1.2 case-data
The liver cancer syndrome of spleen deficiency patient for meeting diagnosis and inclusion criteria is collected in this research from 04 month -2015 years in December, 2014 altogether 11, all cases derive from Hunan Provincial Tumour Hospital's gastroduodenal pancreatic surgery, are made a definite diagnosis through pathologic finding.Patient year Age 24-73 Sui, the median age 56 years old, man 39, female 18, tumor type (poorly differentiated adenocarcinoma 35, medium-low differentiation gland cancer 5 Example, well-differentiated adenocarcinoma 1, middle differentiation gland cancer 2, other types 14).Normal group 45, wherein man 28, female 17 Example, age 23-72 Sui, the median age 52 years old, both from the attached First Hospital physical examination section health of Hunan University of Traditional Chinese Medicine voluntarily Person.The research approach meets human trial ethical standard, and obtains the approval of Ethics Committee, subject it is tested it is preceding Know, and obtain written consent.
1.3 packet design
Liver cancer syndrome of spleen deficiency group VS Normal groups, to reduce experimental error, liver cancer syndrome of spleen deficiency group and Normal group respectively into 2 groups of technologies of row repeat.
1.4 key instruments and reagent consumptive material
1.4.1 key instrument (table 1)
1 key instrument of table
1.4.2 main agents and consumptive material (table 2)
2 main agents of table and consumptive material
1.5 experimental methods and technology
1.5.1 materials
Clinical observation record is carried out according to the special clinical observation table of this subject.Materials evening before that day, clear water was gargled just before going to bed Three times (no longer into any food and drug), using Stimulated whole saliva acquisition mode, from second day morning after gargle before take on an empty stomach Material.It is drawn materials by trained seminar special messenger, the saliva in preceding 5min starts to collect after swallowing naturally, and patient will have been subjected to Containing in entrance, saliva of buccal cavity is gathered to after a certain amount of the sterile roundlet cylindricality cotton of disinfection, which is spat into and is placed in ice bath 15mL saliva centrifuge tubes in, after standing 1h at 4 DEG C, 3000r/min, centrifuge 10min at 4 DEG C, 1mLEP be divided on ice bath Guan Zhong, often 200 μ L of pipe, preserve in -80 DEG C of refrigerator freezings.Sample is taken out during experiment, is thawed on ice, all detection saliva samples Equal 1 freeze thawing.
1.5.2iTRAQ workflow
1.5.2.1 sample process and iTRAQ quantitative experiment flows
As shown in Figure 1：(1) sample protein extracts；(2) protein quantification；(3) proteolysis；(4) iTRAQ is marked；(5) it is high Reversed phase chromatography separation under the conditions of pH；(6) ABI-5600 carries out protein analysis；(7) MASS SPECTRAL DATA ANALYSIS.
1.5.2.2 sample protein extraction process
1. appropriate lysis buffer (7M urea, 2M thiocarbamides, 0.1%CHAPS), vortex mixing are added in sample；
2. ultrasound 60s, 0.2son, 2soff, amplitude 22%；
3. room temperature extracts 30min；
4. 15000r/min, 4 DEG C of centrifugation 20min, carefully take out supernatant；
5. collecting supernatant, frozen after packing packing in -80 DEG C.
1.5.2.3 protein quantification
Using Bradford methods [Marion M.Bradford.Analytical Biochemistry, 1976,72:248- 254] protein concentration of extraction is measured.First mixing the sample with lysis buffer and carrying out certain multiple dilution falls its final concentration to mark In bent range, the sample and standard items (standard protein that BSA is dissolved into series concentration with lysis buffer) that have diluted respectively take 10 μ l are protected from light 20min with 300 μ l protein quantification dyestuffs respectively, and with microplate reader while bioassay standard product and sample are in 595nm Under light absorption value (being shown in Table 1), according to standard items often the relationship of pipe light absorption value and concentration draw standard curve.According to curve equation Calculate each sample protein concentration.
1.5.2.4 proteolysis (Filter Aided Sample Preparation, FASP)
1. 200 μ g protein solutions is taken to be placed in centrifuge tube after protein quantification.
2. 4 μ l reducing agents are added in, 60 DEG C of reaction 1h.
3. add in 2 μ l cysteine blocking agents, room temperature 10min.
4. the protein solution after reductive alkylation is added in the super filter tube of 10K, 12000r/min centrifugation 20min are discarded Collecting pipe bottom solution.
5. adding in 100 μ l, 12000r/min the centrifugation 20min of dissolving buffer solution in iTRAQ kits, collecting pipe bottom is discarded Portion's solution is repeated 3 times (to save reagent, this step uses after can dissolving buffer solution being diluted with water 5 times).
6. the collecting pipe more renewed adds in trypsase in super filter tube, 4 μ g of total amount are (with albumen quality than 1:50), body 50 μ l of product, 37 DEG C of reactions are overnight.
7. next day, 12000r/min centrifugation 20min, peptide fragment solution centrifugation after enzymolysis, digestion is in collecting bottom of the tube.
8. adding in 50 μ l dissolving buffer solutions 5 in super filter tube, 12000r/min centrifuges 20min again, merges with upper step, receives The sample after 100 μ l enzymolysis is obtained in collection bottom of the tube.
1.5.2.5iTRAQ label
1.5.2.5.1iTRAQ labelling experiment step
1. taking out iTRAQ reagents from refrigerator, room temperature is equilibrated to, it willReagent is centrifuged to tube bottom.
2. to every pipe150 μ l isopropanols are added in reagent, vortex oscillation is centrifuged to tube bottom.
3. 50 μ l samples (100 μ g enzymolysis products) is taken to be transferred in new centrifuge tube.
4. iTRAQ reagents are dosed in sample, vortex oscillation, centrifuge to tube bottom, react at room temperature 2h.
5. adding in 100 μ l water terminates reaction.
6. in order to detect labeling effciency and dosing accuracy, 1ul mixing is respectively taken out from 4 groups of samples, with Ziptip desalinations It carries out MALDI-TOF-TOF (ABSCIEX4800Plus) afterwards to identify, confirmation flag reaction is good.
7. the sample after mixed mark, vortex oscillation are centrifuged to tube bottom.
8. vacuum refrigeration centrifugal drying.
9. the sample freezen protective after draining is for use.
1.5.2.5.2 sample label situation (table 3)
3 sample of table marks situation
Number Sample ID Labelled reagent label
1 Normal group -1 113
2 Normal group -2 114
3 Liver cancer syndrome of spleen deficiency group -1 117
4 Liver cancer syndrome of spleen deficiency group -2 118
1.5.2.6 the offline pre-separation of peptide hydrolysis and LC-MS/MS mass spectral analyses
1.5.2.6.1 the reversed phase chromatography separation under the conditions of high pH
(1) allotment of reagent needed for
Mobile phase A：98%ddH2O, 2% acetonitrile (pH10)
Mobile phase B：98% acetonitrile, 2%ddH2O (pH10)
DdH2O ammonium hydroxide tune pH value to 10.
(2) experimental procedure
1. the sample after mixed mark is dissolved with 100ul mobile phase As, 14000r/min centrifugation 20min take supernatant for use.
2. detached (45 DEG C of column temperature, Detection wavelength 214nm), detecting system situation using the 400 μ g BSA digested.
3. take the ready sample loadings of 100ul.
4. flow velocity 0.7ml/min, separation gradient is following (table 4)：
Table 4 detaches gradient for the first time
Time (min) Mobile phase B ratio (%)
0 5
5 8
35 18
62 32
64 95
68 95
72 5
1.5.2.6.2ABI-5600 mass spectrum carries out protein analysis
(1) allotment of reagent needed for
Mobile phase A：100% ultra-pure water, 0.1% formic acid
Mobile phase B：100% acetonitrile, 0.1% formic acid
(2) experimental procedure
1. by 20 μ l2% methanol of the component that high pH reverse phase separations obtain, 0.1% formic acid redissolves.
2. 12000r/min centrifuges 10min, supernatant loading is drawn.
3. 10 μ l of loading volume, take sandwich method loading.
4. load flow rate pump 350nl/min, 15min.
5. detaching flow velocity 350nl/min, separation gradient is following (table 5)：
5 second separation gradient of table
Time (min) Mobile phase B ratio (%)
0 4
5 15
40 25
65 35
70 95
82 95
85 4
90 4
(3) mass spectrometry parameters are set
A) source parameters：
Spray voltage：2.1kv；Capillary temperature：250℃；Ion source：Simple spraying source；DP：100；
b)Full MS：Resolution ratio：70000FWHM；Full scan control targe：1e6；Full scan Max.IT：60ms；Scanning Range：350-1800m/z；
c)dd-MS2：Resolution ratio：17500FWHM；AGC targets：5e6；Maximum IT：70ms；Intensity threshold： 5.00E+03； Breaking method：HCD；NCE：29%；TopN：20.
1.5.2.7 MASS SPECTRAL DATA ANALYSIS
(1) database
Selecting for database is using required species, database annotation completeness and sequence reliability as reference frame. When selecting database, it then follows following principle if biology has been sequenced, directly selects the species database, is given birth to if non-sequencing Object then selects and the mostly concerned major class proteome databases of sample.This uses database uniprot databases.
(2) software is retrieved
The mass spectral analysis of iTRAQ is completed by ABI-5600 types mass spectrum, and the mass spectrum original document of generation is using ABI companies Mating business software Protein Pilot processing.
2. result
This experiment has carried out contrast experiment to liver cancer syndrome of spleen deficiency and Normal group.It is opposite and absolute using isotope labelling Quantitative (iTRAQ) technology carries out identification and quantification analysis to the full histone of liver cancer Patients of Spleen Deficiency saliva, preliminary to obtain liver cancer spleen The protein groups difference expression atlas of asthenic symptoms patient.Quantitative proteomics result of study is shown：It reflects altogether using iTRAQ technologies Fixed 1310 albumen；Compared with Normal group, (1.5 times or more are expressed the significant difference albumen identified in liver cancer syndrome of spleen deficiency Difference) totally 283, wherein upregulated protein 156, down-regulation protein 127.
Differential protein is listed as follows：Upregulated protein is as follows：
1) searching number is Q66K66, and gene annotation is Transmembrane protein 198；
2) searching number is Q9ULD9, and gene annotation is Zinc finger protein 608；
3) searching number is Q8IVF7, and gene annotation is Formin-like protein 3；
4) searching number is O60282, and gene annotation is Kinesin heavy chain isoform 5C；
5) searching number is Q86V2, and gene annotation is Kinesin-like protein KIF27；
6) searching number is P06727, and gene annotation is Apolipoprotein A-IV；
7) searching number is P13473, and gene annotation is Lysosome-associated membrane glycoprotein 2；
8) searching number is P31151, and gene annotation is Protein S100-A7；
9) searching number is O75629, and gene annotation is Protein CREG1；
10) searching number is P35321, gene annotation Cornifin-A；
11) searching number is Q9Y239, and gene annotation is Nucleotide-binding oligomerization domain-containing protein 1；
12) searching number is Q9BRA2, and gene annotation is Thioredoxin domain-containing protein 17；
13) searching number is Q07654, and gene annotation is Trefoil factor 3；
14) searching number is P31949, and gene annotation is Protein S100-A11；
15) searching number is Q96RM1, and gene annotation is Small proline-rich protein 2F；
16) searching number is Q6ZUA9, and gene annotation is Maestro heat-like repeat family member 5；
17) searching number is P68032, gene annotation Actin, alpha cardiac musle 1；
18) searching number is P01598, and gene annotation is Ig kappa chain V-I region EU；
19) searching number is P01708, and gene annotation is Ig lambda chain V-II region BUR；
20) searching number is P83593, and gene annotation is Ig kappa chain V-IV region STH (Fragment)；
21) searching number is P02814, and gene annotation is Submaxillary gland androgen-regulated protein 3B；
22) searching number is Q6P158, and gene annotation is Putative ATP-dependent RNA helicase DH 57；
23) searching number is Q9UBH0, and gene annotation is Interleukin-36receptor antagonist protein；
24) searching number is P49221, and gene annotation is Protein-glutamine gamma- glutamyltransferase 4；
25) searching number is P00480, and gene annotation is Ornithine carbamoyltransferase, mitochondrial；
26) searching number is P68871, and gene annotation is Hemoglobin subunit beta；
27) searching number is P01871, and gene annotation is Ig mu chain C region；
28) searching number is O75038, and gene annotation is 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase eta-2；
29) searching number is P31025, gene annotation Lipocalin-1；
30) searching number is P01612, and gene annotation is Ig kappa chain V-I region Mev；
31) searching number is P19957, gene annotation Elafin；
32) searching number is P08697, gene annotation Alpha-2-antiplasmin；
33) searching number is P32119, gene annotation Peroxiredoxin-2；
34) searching number is Q7L0X2, and gene annotation is Glutamate-rich protein 6；
35) searching number is Q9UL2, and gene annotation is Ras-related protein Rab-21；
36) searching number is P22528, gene annotation Cornifin-B；
37) searching number is Q9Y5Y6, and gene annotation is Suppressor of tumorigenicity 14protein；
38) searching number is P69905, and gene annotation is Hemoglobin subunit alpha；
39) searching number is P01743, and gene annotation is；Ig heavy chain V-I region HG3；
40) searching number is Q6ZP82, and gene annotation is Coiled-coil domain-containing protein 141；
41) searching number is Q01469, and gene annotation is Fatty acid-binding protein, epidermal；
42) searching number is P01860, and gene annotation is Ig gamma-3chain C region；
43) searching number is P09211, and gene annotation is Glutathione S-transferase P；
44) searching number is Q8TED4, and gene annotation is Sugar phosphate exchanger 2；
45) searching number is P04179, and gene annotation is Superoxide dismutase [Mn], mitochondrial；
46) searching number is P01624, and gene annotation is Ig kappa chain V-III region POM；
47) searching number is P1059, gene annotation Thioredoxin；
48) searching number is O95274, and gene annotation is Ly6/PLAUR domain-containing protein 3；
49) searching number is Q9GZN4, and gene annotation is Brain-specific serine protease 4；
50) searching number is Q9ULV4, gene annotation Coronin-1C；
51) searching number is P35270, and gene annotation is Sepiapterin reductase；
52) searching number is Q86SG5, and gene annotation is Protein S100-A7A；
53) searching number is P01764, and gene annotation is Ig heavy chain V-III region 23；
54) searching number is P06870, gene annotation Kallikein-1；
55) searching number is Q96PD5, and gene annotation is N-acetylmuramoyl-L-alanine amidase；
56) searching number is Q9UHY7, and gene annotation is Enolase-phosphatase E1；
57) searching number is Q3ZCW2, and gene annotation is Galectin-related protein；
60) searching number is Q92692, gene annotation Nectin-2；
61) searching number is P01834, and gene annotation is Ig kappa chain C region；
62) searching number is P00915, and gene annotation is Carbonic anhydrase 1；
63) searching number is P01771, and gene annotation is Ig heavy chain V-III region HIL；
64) searching number is P01857, and gene annotation is Ig gamma-1chain C region；
65) searching number is P04220, and gene annotation is Ig mu heavy chain disease protein；
66) searching number is P00450, gene annotation Ceruloplasmin；
67) searching number is P00734, gene annotation Prothrombin；
68) searching number is Q6MZM9, and gene annotation is Proline-rich protein 27；
69) searching number is P01610, and gene annotation is Ig kappa chain V-I region WEA；
70) searching number is P02763, and gene annotation is Alpha-1-acid glycoprotein 1；
71) searching number is P15328, and gene annotation is Folate receptor alpha；
72) searching number is P02042, and gene annotation is Hemoglobin subunit delta；
73) searching number is O60701, and gene annotation is UDP-glucose 6-dehydrogenase；
74) searching number is O00154, and gene annotation is Cytosolic acyl coenzyme A thioester hydrolase；
75) searching number is P41439, and gene annotation is Folate receptor gamma；
76) searching number is Q14126, gene annotation Desmoglein-2；
77) searching number is Q92734, and gene annotation is Protein TFG；
78) searching number is Q92876, gene annotation Kallikrein-6；
79) searching number is O60635, gene annotation Tetraspanin-1；
80) searching number is P19404, and gene annotation is NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial ；
81) searching number is P02787, gene annotation Serotransferrin；
82) searching number is P22532, and gene annotation is Small proline-rich protein 2D；
83) searching number is B3EWG3, and gene annotation is Protein FAM25A；
84) searching number is P68402, and gene annotation is Platelet-activating factor acetylhydrolase IB subunit beta；
85) searching number is O75506, and gene annotation is Heat shock factor-binding protein 1；
86) searching number is P01770, and gene annotation is Ig heavy chain V-III region NIE；
87) searching number is Q8IVW1, and gene annotation is ADP-ribosylation factor-like protein 17；
88) searching number is P01040, gene annotation Cystatin-A；
89) searching number is P04217, gene annotation Alpha-1B-glycoprotein；
90) searching number is P04181, and gene annotation is Ornithine aminotransferase, mitochondrial ；
91) searching number is P05388, and gene annotation is 60S acidic ribosomal protein P0；
92) searching number is Q15223, gene annotation Nectin-1；
93) searching number is P0C870, and gene annotation is JmjC domain-containing protein 7；
94) searching number is P20160, gene annotation Azurocidin；
95) searching number is P02768, and gene annotation is Serum albumin；
96) searching number is O15231, and gene annotation is Zinc finger protein 185；
97) searching number is Q96PF2, and gene annotation is Testis-specific serine/threonine-protein kinase 2；
98) searching number is Q8NI51, and gene annotation is Transcriptional repressor CTCFL；
99) searching number is P01859, and gene annotation is Ig gamma-2chain C region；
100) searching number is P02760, and gene annotation is Protein AMBP；
101) searching number is P08236, gene annotation Beta-glucuronidase；
102) searching number is Q9Y3D6, and gene annotation is Mitochondrial fission 1protein；
103) searching number is P80188, and gene annotation is Neutrophil gelatinase-associated lipocalin；
104) searching number is Q6UW32, and gene annotation is Insulin growth factor-like family member 1；
105) searching number is O75131, gene annotation Copine-3；
106) searching number is Q04760, and gene annotation is Lactoylglutathione lyase；
107) searching number is Q9BPV8, and gene annotation is P2Y purinoceptor 13；
108) searching number is P58062, and gene annotation is Serine protease inhibitor Kazal-type 7；
109) searching number is P02749, and gene annotation is Beta-2-glycoprotein 1；
110) searching number is P02774, and gene annotation is Vitamin D-binding protein；
111) searching number is P01762, and gene annotation is Ig heavy chain V-III region TRO；
112) searching number is P00738, gene annotation Haptoglobin；
113) searching number is P01767, and gene annotation is Ig heavy chain V-III region BUT；
114) searching number is P02671, and gene annotation is Fibrinogen alpha chain；
115) searching number is P04003, and gene annotation is C4b-binding protein alpha chain；
116) searching number is Q96L46, and gene annotation is Calpain small subunit 2；
117) searching number is Q9NZH8, gene annotation Interleukin-36gamma；
118) searching number is P31944, gene annotation Caspase-14；
119) searching number is P02753, and gene annotation is Retinol-binding protein 4；
120) searching number is Q13740, gene annotation CD166antigen；
121) searching number is Q8WWY7, and gene annotation is WAP four-disulfide core domain protein 12；
122) searching number is O75116, and gene annotation is Rho-associated protein kinase 2；
123) searching number is Q14210, and gene annotation is Lymphocyte antigen 6D；
124) searching number is P0CG39, and gene annotation is POTE ankyrin domain family member J；
125) searching number is Q9UKR3, gene annotation Kallikrein-13；
126) searching number is P01605, and gene annotation is Ig kappa chain V-I region Lay；
127) searching number is P01778, and gene annotation is Ig heavy chain V-III region ZAP；
128) searching number is P51159, and gene annotation is Ras-related protein Rab-27A；
129) searching number is P02790, gene annotation Hemopexin；
130) searching number is P01781, and gene annotation is Ig heavy chain V-III region GAL；
131) searching number is P17516, and gene annotation is Aldo-keto reductase family 1member C4；
132) searching number is P06731, and gene annotation is Carcinoembryonic antigen-related cell adhesion molecule 5；
133) searching number is P05155, and gene annotation is Plasma protease C1inhibitor；
134) searching number is P28799, gene annotation Granulins；
135) searching number is Q02487, gene annotation Desmocollin-2；
136) searching number is Q96AT9, and gene annotation is Ribulose-phosphate 3-epimerase；
137) searching number is O00748, and gene annotation is Cocaine esterase；
138) searching number is P29508, and gene annotation is Serpin B3；
139) searching number is Q14CN2, and gene annotation is Calcium-activated chloride channel regulator 4；
140) searching number is P0C0L5, and gene annotation is Complement C4-B；
141) searching number is P01776, and gene annotation is Ig heavy chain V-III region WAS；
142) searching number is P12830, gene annotation Cadherin-1；
143) searching number is Q14574, gene annotation Desmocollin-3；
144) searching number is P01623, and gene annotation is Ig kappa chain V-III region WOL；
145) searching number is Q8N271, gene annotation Prominin-2；
146) searching number is P01601, and gene annotation is Ig kappa chain V-ID region 16 (Fragment)；
147) searching number is Q12792, gene annotation Twinfilin-1；
148) searching number is P69891, and gene annotation is Hemoglobin subunit gamma-1；
149) searching number is P30048, and gene annotation is Thioredoxin-dependent peroxide reductase,mitochondrial；
150) searching number is P08603, and gene annotation is Complement factor H；
151) searching number is P02788, gene annotation Lactotransferrin；
152) searching number is O75347, and gene annotation is Tubulin-specific chaperone A；
153) searching number is P01023, gene annotation Alpha-2-macroglobulin；
154) searching number is P06748, gene annotation Nucleophosmin；
155) searching number is P01833, and gene annotation is Polymeric immunoglobulin receptor；
156) searching number is P04632, and gene annotation is Calpain small subunit 1；
157) searching number is P24158, gene annotation Myeloblastin；
158) searching number is P19971, and gene annotation is Thymidine phosphorylase；
Down-regulation protein is as follows：
1) searching number is Q12955, gene annotation Ankyrin-3；
2) searching number is O95716, and gene annotation is Ras-related protein Rab-3D；
3) searching number is Q2KHT4, and gene annotation is Germ cell-specific gene 1protein；
4) searching number is Q13043, and gene annotation is Serine/threonine-protein kinase 4；
5) searching number is F5H4A9, and gene annotation is Uncharacterized membrane protein C3orf80；
6) searching number is Q8IWB9, and gene annotation is Testis-expressed sequence 2protein；
7) searching number is P01602, and gene annotation is Ig heavy chain V-I region 5 (Fragment)；
8) searching number is Q8TC84, and gene annotation is Fibronectin type 3and ankyrin repeat domains protein 1；
9) searching number is Q8IV20, and gene annotation is Laccase domain-containing protein 1；
10) searching number is P08754, and gene annotation is Guanine nucleotide-binding protein G (k) subunit alpha；
11) searching number is Q6T4P5, and gene annotation is Lipid phosphate phosphatase-related protein type 3；
12) searching number is P50748, and gene annotation is Kinetochore-associated protein 1；
13) searching number is Q8IVL1, and gene annotation is Neuron navigator 2；
14) searching number is P60953, and gene annotation is Cell division control protein 42homolog；
15) searching number is P02808, gene annotation Statherin；
16) searching number is Q9UKK3, and gene annotation is Poly [ADP-ribose] polymerase 4；
17) searching number is P78371, and gene annotation is T-complex protein 1subunit beta；
18) searching number is O00602, gene annotation Ficolin-1；
19) searching number is P22392, and gene annotation is Nucleoside diphosphate kinase B；
20) searching number is Q9BRZ2, and gene annotation is E3ubiquitin-protein ligase TRIM56；
21) searching number is O75015, and gene annotation is Low affinity immunoglobulin gamma Fc region receptor III-B；
22) searching number is Q15493, gene annotation Regucalcin；
23) searching number is P01703, and gene annotation is Ig lambda chainV-I regionNEWM；
24) searching number is P16401, and gene annotation is Histone H1.5；
25) searching number is P80303, gene annotation Nucleobindin-2；
26) searching number is P32243, and gene annotation is Homeobox protein OTX2；
27) searching number is Q16378, and gene annotation is Proline-rich protein 4；
28) searching number is Q7Z5M8, and gene annotation is Abhydrolase domain-containing protein 12B；
29) searching number is P58499, and gene annotation is Protein FAM3B；
30) searching number is P42765, and gene annotation is 3-ketoacyl-CoA thiolase, mitochondrial；
31) searching number is Q9P0M2, and gene annotation is A-kinase anchor protein 7isoform gamma；
32) searching number is P22626, and gene annotation is Heterogeneous nuclear ribonucleoproteins A2/B1；
33) searching number is Q9UNU6, and gene annotation is 7-alpha-hydroxycholest-4-en-3-one 12- alpha-hydroxylase；
34) searching number is P20929, gene annotation Nebulin；
35) searching number is P04259, gene annotation Keratin, type II cytoskeletal 6B；
36) searching number is P04438, and gene annotation is Ig heavy chain V-II region SESS；
37) searching number is O43852, gene annotation Calumenin；
38) searching number is Q9UHY1, and gene annotation is Nuclear receptor-binding protein；
39) searching number is Q12841, and gene annotation is Follistatin-related protein 1；
40) searching number is Q9UPT6, and gene annotation is C-Jun-amino-terminal kinase-interacting protein 3；
41) searching number is Q06828, gene annotation Fibromodulin；
42) searching number is Q9H4M9, and gene annotation is EH domain-containing protein 1；
43) searching number is Q04917 14-3-3, and gene annotation is protein eta；
44) searching number is Q9HCU9, and gene annotation is Breast cancer metastasis-suppressor 1；
45) searching number is P05089, gene annotation Arginase-1；
46) searching number is Q96RS0, and gene annotation is Trimethylguanosine synthase；
47) searching number is Q8IWF6, and gene annotation is Protein DENND6A；
48) searching number is Q92521, and gene annotation is GPI mannosyltransferase 3；
49) searching number is P33241, and gene annotation is Lymphocyte-specific protein 1；
50) searching number is Q96FZ7, and gene annotation is Charged multivesicular body protein 6；
51) searching number is Q9ULZ3, and gene annotation is Apoptosis-associated speck-like protein containing a CARD；
52) searching number is P25787, and gene annotation is Proteasome subunitalpha type-2；
53) searching number is O60361, and gene annotation is Putative nucleoside diphosphate kinase；
54) searching number is Q8TAX7, gene annotation Mucin-7；
55) searching number is Q92688, and gene annotation is Acidic leucine-richnuclear phosphoprotein 32 family member B；
56) searching number is P40394, and gene annotation is Alcohol dehydrogenase class 4mu/sigma chain；
57) searching number is Q14289, and gene annotation is Protein-tyrosine kinase 2-beta；
58) searching number is P55008, and gene annotation is Allograft inflammatory factor 1；
59) searching number is P08571, and gene annotation is Monocyte differentiationantigenCD14；
60) searching number is P21439, and gene annotation is Multidrugresistance protein3；
61) searching number is O95777U6, and gene annotation is snRNA-associated Sm-like protein LSm8；
62) searching number is Q96DA0, and gene annotation is Zymogen granule protein16homologB；
63) searching number is Q16651, gene annotation Prostasin；
64) searching number is Q9Y4Y9, and gene annotation is U6snRNA-associated Sm-like protein LSm5；
65) searching number is P26583, and gene annotation is Highmobility group proteinB2；
66) searching number is Q9H008, and gene annotation is Phospholysine phosphohistidine inorganic pyrophosphate phosphatase；
67) searching number is P01742, and gene annotation is Igheavy chainV-I regionEU；
68) searching number is Q15631, gene annotation Translin；
69) searching number is Q9HC38, and gene annotation is Glyoxalase domain-containing protein4；
70) searching number is Q9Y376, and gene annotation is Calcium-binding protein39；
71) searching number is Q96DR5, and gene annotation is BPI fold-containing family Amember 2；
72) searching number is P07477Trypsin-1；
73) searching number is Q96C19, and gene annotation is EF-hand domain-containing protein D2；
74) searching number is P16870, and gene annotation is Carboxypeptidase E；
75) searching number is Q8NI35, and gene annotation is InaD-like protein；
76) searching number is P05362, and gene annotation is Intercellular adhesionmolecule 1；
77) searching number is P15374, and gene annotation is Ubiquitin carboxyl-terminalhydrolase isozyme L3；
78) searching number is Q6ZNW5, and gene annotation is GDP-D-glucose phosphorylase 1；
79) searching number is Q15370, and gene annotation is Transcriptionelongationfactor B polypeptide 2；
80) searching number is P0CG05, and gene annotation is Iglambda-2chain C regions；
81) searching number is P30740, and gene annotation is Leukocyte elastase inhibitor；
82) searching number is Q7Z6J4, gene annotation FYVE, RhoGEF and PH domain-containing protein 2；
83) searching number is Q9H7L9, and gene annotation is Sin3histone deacetylase corepressor complex component SDS3；
84) searching number is P00533, and gene annotation is Epidermal growthfactor receptor；
85) searching number is P30046, and gene annotation is D-dopachrome decarboxylase；
86) searching number is Q96BQ1, gene annotation ProteinFAM3D；
87) searching number is Q9NP98, gene annotation Myozenin-1；
88) searching number is P31146, gene annotation Coronin-1A；
89) searching number is O75503, and gene annotation is Ceroid-lipofuscinosis neuronal protein5；
90) searching number is P63279, and gene annotation is SUMO-conjugatingenzyme UBC9；
91) searching number is Q8IUE6, and gene annotation is Histone H2Atype 2-B；
92) searching number is Q9NZ53, and gene annotation is Podocalyxin-like protein 2；
93) searching number is Q8NF91, gene annotation Nesprin-1；
94) searching number is Q9BS40, gene annotation Latexin；
95) searching number is P09382, gene annotation Galectin-1；
96) searching number is Q9BRF8, gene annotation Serine/threonine-proteinphosphatase CPPED1；
97) searching number is P08579, and gene annotation is U2small nuclear ribonucleoproteinB "；
98) searching number is P49720, and gene annotation is Proteasome subunitbeta type-3；
99) searching number is P52566, and gene annotation is Rho GDP-dissociationinhibitor 2；
100) searching number is Q8NC60, and gene annotation is Nitric oxide-associated protein1；
101) searching number is P10909, gene annotation Clusterin；
102) searching number is P01712, and gene annotation is glambda chain V-II regionWIN；
103) searching number is P23280, and gene annotation is Carbonic anhydrase 6；
104) searching number is Q8IWQ3, and gene annotation is Serine/threonine-protein kinase BRSK2；
105) searching number is Q86VR7, and gene annotation is V-set and immunoglobulin domain- containing protein 10-like；
106) searching number is Q99439, gene annotation Calponin-2；
107) searching number is Q9GZZ8, and gene annotation is Extracellular glycoproteinlacritin；
108) searching number is O43707, gene annotation Alpha-actinin-4；
109) searching number is P62253, and gene annotation is Ubiquitin-conjugating enzyme E2G1；
110) searching number is Q13421, gene annotation Mesothelin；
111) searching number is P35237, and gene annotation is Serpin B6；
112) searching number is P14550, and gene annotation is Alcohol dehydrogenase [NADP (+)]；
113) searching number is P06737, and gene annotation is Glycogen phosphorylase, liver form；
114) searching number is O00584, and gene annotation is Ribonuclease T2；
115) searching number is P62993, and gene annotation is Growth factor receptor-bound protein 2；
116) searching number is P01707, and gene annotation is Ig lambda chain V-II region TRO；
117) searching number is P01036, gene annotation Cystatin-S；
118) searching number is P35908, gene annotation Keratin, type II cytoskeletal 2epidermal；
119) searching number is O95684, and gene annotation is FGFR1oncogene partner；
120) searching number is O75891, and gene annotation is Cytosolic 10-formyltetrahydrofolate dehydrogenase；
121) searching number is Q86VD1, and gene annotation is MORC family CW-type zinc finger protein 1；
122) searching number is Q9Y490, gene annotation Talin-1；
123) searching number is P05154, and gene annotation is Plasma serine protease inhibitor；
124) searching number is O15143, and gene annotation is Actin-related protein 2/3complex subunit 1B；
125) searching number is O75563, and gene annotation is Src kinase-associated phosphoprotein 2；
126) searching number is A0M8Q6, and gene annotation is Ig lambda-7chain C region；
127) searching number is Q6JBY9, and gene annotation is CapZ-interacting protein.
Plurality of differential protein (such as Cadherin-1 (CDH1), Cell division control protein 42 homolog(CDC42)、Growth factor receptor-bound protein 2(GRB2)、Ankyrin-3(ANK3)、 Guanine nucleotide-binding protein G (k) subunit alpha (GNAI3) etc.) to take part in some important Metabolic pathway, may be closely related with the generation, development, prognosis of liver cancer.
ITRAQ labelling techniques can detach and identify simultaneously Thousands of protein, can farthest reflect protein group Information.Using the research of iTRAQ quantitative proteomics, we have found multiple liver cancer syndrome of spleen deficiency early stages that may be valuable Saliva hurtless measure molecular diagnostics biological marker；Further disclose the material base of liver cancer syndrome of spleen deficiency molecular group level with Scientific meaning is laid a good foundation for its further occurrence and development mechanism and its biomarker research.
The present invention can quick, reliable proteomic techniques scheme find the specific proteins in liver cancer syndrome of spleen deficiency Marker is that the early stage diagnosis and treatment of liver cancer syndrome of spleen deficiency, postoperative recurrence, transfer and Observation On The Prognosis establish a kind of noninvasive, easy, quick reality Check and evaluation means.
The better embodiment of this patent is explained in detail above, but this patent is not limited to above-mentioned embodiment party Formula, can also be under the premise of this patent objective not be departed from the knowledge that one skilled in the relevant art has Various changes can be made.

Claims (1)

1. the method for the liver cancer syndrome of spleen deficiency biological marker analyte detection based on sialoprotein matter group, which is characterized in that specific steps It is as follows：
(1) sample protein extracts：The lysis buffer that 5% is added in sample carries out vortex mixing, ultrasonic 60s, 22% Room of amplitude Temperature extraction 30min；15000r/min, 4 DEG C of centrifugation 20min, take out supernatant；Supernatant is collected, is frozen after packing packing in -80 DEG C；
(2) protein quantification：The protein concentration of extraction is measured using Bradford methods, lysis buffer is first mixed the sample with and carries out centainly Multiple dilution its final concentration is made to fall in the range of mark song, the sample and standard items diluted respectively take 10 μ l respectively with 300 μ l albumen Quantitative stain is protected from light 20min, with microplate reader simultaneously the light absorption value of bioassay standard product and sample under 595nm, according to standard Often the relationship of pipe light absorption value and concentration draws standard curve to product, and each sample protein concentration is calculated according to curve equation；
(3) proteolysis：200 μ g protein solutions is taken to be placed in centrifuge tube after protein quantification；Add in 4 μ l reducing agents, 60 DEG C of reactions 1h；The cysteine blocking agent of 2 μ l is added in, reacts at room temperature 10min；Protein solution after reductive alkylation is added in into the super of 10K In chimney filter, 12000r/min centrifugation 20min discard collecting pipe bottom solution；Add in the solution buffer solution in iTRAQ kits 100 μ l, 12000 leave heart 20min, discard collecting pipe bottom solution, are repeated 3 times；The collecting pipe more renewed adds in super filter tube Enter trypsase, 4 μ g of total amount, 50 μ l of volume, 37 DEG C of reactions are overnight；Next day, 12000r/min centrifugation 20min, after enzymolysis, digestion Peptide fragment solution centrifugation in collect bottom of the tube；50 μ l dissolving buffer solutions are added in super filter tube, 12000r/min is centrifuged again 20min merges with upper step, collects the sample that bottom of the tube is obtained after 100 μ l enzymolysis；
(4) iTRAQ is marked：ITRAQ reagents are taken out from refrigerator, room temperature is equilibrated to, iTRAQ reagents is centrifuged to tube bottom；To every 150 μ l isopropanols are added in pipe iTRAQ reagents, vortex oscillation is centrifuged to tube bottom；The sample after 50 μ l enzymolysis is taken to be transferred to new In centrifuge tube；ITRAQ reagents are added in sample, vortex oscillation, centrifuge to tube bottom, react at room temperature 2h；Add in 100 μ l water end Only react；1ul mixing is respectively taken out from 4 groups of samples, with carrying out MALDI-TOF-TOF identifications, confirmation flag after Ziptip desalinations Reaction is good；Sample after mixed mark, vortex oscillation are centrifuged to tube bottom；Vacuum refrigeration centrifugal drying；Sample after draining is cold Freeze and preserve for use；
(5) reversed phase chromatography separation under the conditions of high pH：The allotment of required reagent：Mobile phase A：98%ddH2O, 2% acetonitrile, pH10；Mobile phase B：98% acetonitrile, 2%ddH2O, pH10；DdH2O ammonium hydroxide tune pH value to 10；Sample after mixed mark is used 100ul mobile phase As dissolve, and 14000r/min centrifugation 20min take supernatant for use；It is detached using the 400 μ g BSA digested, Detecting system situation；Take the ready sample loadings of 100ul；Flow velocity 0.7ml/min obtains separation gradient data；
(6) ABI-5600 carries out protein analysis：The allotment of required reagent：Mobile phase A：100% ultra-pure water, 0.1% formic acid；Stream Dynamic phase B：100% acetonitrile, 0.1% formic acid；By 20 μ l2% methanol of the component that high pH reverse phase separations obtain, 0.1% formic acid is answered It is molten；12000r/min centrifuges 10min, draws supernatant loading, and 10 μ l of loading volume take sandwich method loading；Load flow rate pump 350nl/min, 15min；Flow velocity 350nl/min is detached, obtains separation gradient data；
(7) MASS SPECTRAL DATA ANALYSIS：When selecting database, if biology has been sequenced, the species database is directly selected, if Non- sequencing biology, then select and the mostly concerned major class proteome databases of sample；Using ABI-5600 type mass spectrographs Carry out the mass spectral analysis of iTRAQ；
(8) testing result：Contrast experiment is carried out to liver cancer syndrome of spleen deficiency and Normal group, quantitative proteomics result of study is shown Show：Identify 1297 albumen；Compared with Normal group, 1.5 times or more differential expressions being identified in liver cancer syndrome of spleen deficiency it is aobvious Write differential protein totally 140, wherein upregulated protein 64, down-regulation protein 76；Differential protein is listed as follows：Upregulated protein is such as Under：
1) searching number is Q66K66, and gene annotation is Transmembrane protein 198；
2) searching number is Q9ULD9, and gene annotation is Zinc finger protein 608；
3) searching number is Q8IVF7, and gene annotation is Formin-like protein 3；
4) searching number is O60282, and gene annotation is Kinesin heavy chain isoform 5C；
5) searching number is Q86V2, and gene annotation is Kinesin-like protein KIF27；
6) searching number is P06727, and gene annotation is Apolipoprotein A-IV；
7) searching number is P13473, and gene annotation is Lysosome-associated membrane glycoprotein 2；
8) searching number is P31151, and gene annotation is Protein S100-A7；
9) searching number is O75629, and gene annotation is Protein CREG1；
10) searching number is P35321, gene annotation Cornifin-A；
11) searching number is Q9Y239, and gene annotation is Nucleotide-binding oligomerization domain- containing protein 1；
12) searching number is Q9BRA2, and gene annotation is Thioredoxin domain-containing protein 17；
13) searching number is Q07654, and gene annotation is Trefoil factor 3；
14) searching number is P31949, and gene annotation is Protein S100-A11；
15) searching number is Q96RM1, and gene annotation is Small proline-rich protein 2F；
16) searching number is Q6ZUA9, and gene annotation is Maestro heat-like repeat family member 5；
17) searching number is P68032, gene annotation Actin, alpha cardiac musle 1；
18) searching number is P01598, and gene annotation is Ig kappa chain V-I region EU；
19) searching number is P01708, and gene annotation is Ig lambda chain V-II region BUR；
20) searching number is P83593, and gene annotation is Ig kappa chain V-IV region STH (Fragment)；
21) searching number is P02814, and gene annotation is Submaxillary gland androgen-regulated protein 3B；
22) searching number is Q6P158, and gene annotation is Putative ATP-dependent RNAhelicase DH57；
23) searching number is Q9UBH0, and gene annotation is Interleukin-36receptor antagonist protein；
24) searching number is P49221, and gene annotation is Protein-glutamine gamma-glutamyltransferase 4；
25) searching number is P00480, and gene annotation is Ornithine carbamoyltransferase, mitochondrial；
26) searching number is P68871, and gene annotation is Hemoglobin subunit beta；
27) searching number is P01871, and gene annotation is Ig mu chain C region；
28) searching number is O75038, and gene annotation is 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase eta-2；
29) searching number is P31025, gene annotation Lipocalin-1；
30) searching number is P01612, and gene annotation is Ig kappa chain V-I region Mev；
31) searching number is P19957, gene annotation Elafin；
32) searching number is P08697, gene annotation Alpha-2-antiplasmin；
33) searching number is P32119, gene annotation Peroxiredoxin-2；
34) searching number is Q7L0X2, and gene annotation is Glutamate-rich protein 6；
35) searching number is Q9UL2, and gene annotation is Ras-related protein Rab-21；
36) searching number is P22528, gene annotation Cornifin-B；
37) searching number is Q9Y5Y6, and gene annotation is Suppressor of tumorigenicity 14protein；
38) searching number is P69905, and gene annotation is Hemoglobin subunit alpha；
39) searching number is P01743, and gene annotation is；Ig heavy chain V-I region HG3；
40) searching number is Q6ZP82, and gene annotation is Coiled-coil domain-containing protein 141；
41) searching number is Q01469, and gene annotation is Fatty acid-binding protein, epidermal；
42) searching number is P01860, and gene annotation is Ig gamma-3chain C region；
43) searching number is P09211, and gene annotation is Glutathione S-transferase P；
44) searching number is Q8TED4, and gene annotation is Sugar phosphate exchanger 2；
45) searching number is P04179, and gene annotation is Superoxide dismutase [Mn], mitochondrial；
46) searching number is P01624, and gene annotation is Ig kappa chain V-III region POM；
47) searching number is P1059, gene annotation Thioredoxin；
48) searching number is O95274, and gene annotation is Ly6/PLAUR domain-containing protein 3；
49) searching number is Q9GZN4, and gene annotation is Brain-specific serine protease 4；
50) searching number is Q9ULV4, gene annotation Coronin-1C；
51) searching number is P35270, and gene annotation is Sepiapterin reductase；
52) searching number is Q86SG5, and gene annotation is Protein S100-A7A；
53) searching number is P01764, and gene annotation is Ig heavy chain V-III region 23；
54) searching number is P06870, gene annotation Kallikein-1；
55) searching number is Q96PD5, and gene annotation is N-acetylmuramoyl-L-alanine amidase；
56) searching number is Q9UHY7, and gene annotation is Enolase-phosphatase E1；
57) searching number is Q3ZCW2, and gene annotation is Galectin-related protein；
60) searching number is Q92692, gene annotation Nectin-2；
61) searching number is P01834, and gene annotation is Ig kappa chain C region；
62) searching number is P00915, and gene annotation is Carbonic anhydrase 1；
63) searching number is P01771, and gene annotation is Ig heavy chain V-III region HIL；
64) searching number is P01857, and gene annotation is Ig gamma-1chain C region；
65) searching number is P04220, and gene annotation is Ig mu heavy chain disease protein；
66) searching number is P00450, gene annotation Ceruloplasmin；
67) searching number is P00734, gene annotation Prothrombin；
68) searching number is Q6MZM9, and gene annotation is Proline-rich protein 27；
69) searching number is P01610, and gene annotation is Ig kappa chain V-I region WEA；
70) searching number is P02763, and gene annotation is Alpha-1-acid glycoprotein 1；
71) searching number is P15328, and gene annotation is Folate receptor alpha；
72) searching number is P02042, and gene annotation is Hemoglobin subunit delta；
73) searching number is O60701, and gene annotation is UDP-glucose 6-dehydrogenase；
74) searching number is O00154, and gene annotation is Cytosolic acyl coenzyme A thioester hydrolase；
75) searching number is P41439, and gene annotation is Folate receptor gamma；
76) searching number is Q14126, gene annotation Desmoglein-2；
77) searching number is Q92734, and gene annotation is Protein TFG；
78) searching number is Q92876, gene annotation Kallikrein-6；
79) searching number is O60635, gene annotation Tetraspanin-1；
80) searching number is P19404, and gene annotation is NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial；
81) searching number is P02787, gene annotation Serotransferrin；
82) searching number is P22532, and gene annotation is Small proline-rich protein 2D；
83) searching number is B3EWG3, and gene annotation is Protein FAM25A；
84) searching number is P68402, and gene annotation is Platelet-activating factor acetylhydrolase IB subunit beta；
85) searching number is O75506, and gene annotation is Heat shock factor-binding protein 1；
86) searching number is P01770, and gene annotation is Ig heavy chain V-III region NIE；
87) searching number is Q8IVW1, and gene annotation is ADP-ribosylation factor-like protein 17；
88) searching number is P01040, gene annotation Cystatin-A；
89) searching number is P04217, gene annotation Alpha-1B-glycoprotein；
90) searching number is P04181, and gene annotation is Ornithine aminotransferase, mitochondrial；
91) searching number is P05388, and gene annotation is 60S acidic ribosomal protein P0；
92) searching number is Q15223, gene annotation Nectin-1；
93) searching number is P0C870, and gene annotation is JmjC domain-containing protein 7；
94) searching number is P20160, gene annotation Azurocidin；
95) searching number is P02768, and gene annotation is Serum albumin；
96) searching number is O15231, and gene annotation is Zinc finger protein 185；
97) searching number is Q96PF2, and gene annotation is Testis-specific serine/threonine-protein kinase 2；
98) searching number is Q8NI51, and gene annotation is Transcriptional repressor CTCFL；
99) searching number is P01859, and gene annotation is Ig gamma-2chain C region；
100) searching number is P02760, and gene annotation is Protein AMBP；
101) searching number is P08236, gene annotation Beta-glucuronidase；
102) searching number is Q9Y3D6, and gene annotation is Mitochondrial fission 1protein；
103) searching number is P80188, and gene annotation is Neutrophil gelatinase-associated lipocalin；
104) searching number is Q6UW32, and gene annotation is Insulin growth factor-like family member 1；
105) searching number is O75131, gene annotation Copine-3；
106) searching number is Q04760, and gene annotation is Lactoylglutathione lyase；
107) searching number is Q9BPV8, and gene annotation is P2Y purinoceptor 13；
108) searching number is P58062, and gene annotation is Serine protease inhibitor Kazal-type 7；
109) searching number is P02749, and gene annotation is Beta-2-glycoprotein 1；
110) searching number is P02774, and gene annotation is Vitamin D-binding protein；
111) searching number is P01762, and gene annotation is Ig heavy chain V-III region TRO；
112) searching number is P00738, gene annotation Haptoglobin；
113) searching number is P01767, and gene annotation is Ig heavy chain V-III region BUT；
114) searching number is P02671, and gene annotation is Fibrinogen alpha chain；
115) searching number is P04003, and gene annotation is C4b-binding protein alpha chain；
116) searching number is Q96L46, and gene annotation is Calpain small subunit 2；
117) searching number is Q9NZH8, gene annotation Interleukin-36gamma；
118) searching number is P31944, gene annotation Caspase-14；
119) searching number is P02753, and gene annotation is Retinol-binding protein 4；
120) searching number is Q13740, gene annotation CD166antigen；
121) searching number is Q8WWY7, and gene annotation is WAP four-disulfide core domain protein 12；
122) searching number is O75116, and gene annotation is Rho-associated protein kinase 2；
123) searching number is Q14210, and gene annotation is Lymphocyte antigen 6D；
124) searching number is P0CG39, and gene annotation is POTE ankyrin domain family member J；
125) searching number is Q9UKR3, gene annotation Kallikrein-13；
126) searching number is P01605, and gene annotation is Ig kappa chain V-I region Lay；
127) searching number is P01778, and gene annotation is Ig heavy chain V-III region ZAP；
128) searching number is P51159, and gene annotation is Ras-related protein Rab-27A；
129) searching number is P02790, gene annotation Hemopexin；
130) searching number is P01781, and gene annotation is Ig heavy chain V-III region GAL；
131) searching number is P17516, and gene annotation is Aldo-keto reductase family 1member C4；
132) searching number is P06731, and gene annotation is Carcinoembryonic antigen-related cell adhesion molecule 5；
133) searching number is P05155, and gene annotation is Plasma protease C1inhibitor；
134) searching number is P28799, gene annotation Granulins；
135) searching number is Q02487, gene annotation Desmocollin-2；
136) searching number is Q96AT9, and gene annotation is Ribulose-phosphate 3-epimerase；
137) searching number is O00748, and gene annotation is Cocaine esterase；
138) searching number is P29508, and gene annotation is Serpin B3；
139) searching number is Q14CN2, and gene annotation is Calcium-activated chloride channel regulator 4；
140) searching number is P0C0L5, and gene annotation is Complement C4-B；
141) searching number is P01776, and gene annotation is Ig heavy chain V-III region WAS；
142) searching number is P12830, gene annotation Cadherin-1；
143) searching number is Q14574, gene annotation Desmocollin-3；
144) searching number is P01623, and gene annotation is Ig kappa chain V-III region WOL；
145) searching number is Q8N271, gene annotation Prominin-2；
146) searching number is P01601, and gene annotation is Ig kappa chain V-ID region 16 (Fragment)；
147) searching number is Q12792, gene annotation Twinfilin-1；
148) searching number is P69891, and gene annotation is Hemoglobin subunit gamma-1；
149) searching number is P30048, and gene annotation is Thioredoxin-dependent peroxide reductase, mitochondrial；
150) searching number is P08603, and gene annotation is Complement factor H；
151) searching number is P02788, gene annotation Lactotransferrin；
152) searching number is O75347, and gene annotation is Tubulin-specific chaperone A；
153) searching number is P01023, gene annotation Alpha-2-macroglobulin；
154) searching number is P06748, gene annotation Nucleophosmin；
155) searching number is P01833, and gene annotation is Polymeric immunoglobulin receptor；
156) searching number is P04632, and gene annotation is Calpain small subunit 1；
157) searching number is P24158, gene annotation Myeloblastin；
158) searching number is P19971, and gene annotation is Thymidine phosphorylase；
Down-regulation protein is as follows：
1) searching number is Q12955, gene annotation Ankyrin-3；
2) searching number is O95716, and gene annotation is Ras-related protein Rab-3D；
3) searching number is Q2KHT4, and gene annotation is Germ cell-specific gene 1protein；
4) searching number is Q13043, and gene annotation is Serine/threonine-protein kinase 4；
5) searching number is F5H4A9, and gene annotation is Uncharacterized membrane protein C3orf80；
6) searching number is Q8IWB9, and gene annotation is Testis-expressed sequence 2protein；
7) searching number is P01602, and gene annotation is Ig heavy chain V-I region 5 (Fragment)；
8) searching number is Q8TC84, and gene annotation is Fibronectin type 3and ankyrin repeat domains protein 1；
9) searching number is Q8IV20, and gene annotation is Laccase domain-containing protein 1；
10) searching number is P08754, and gene annotation is Guanine nucleotide-binding protein G (k) subunit alpha；
11) searching number is Q6T4P5, and gene annotation is Lipid phosphate phosphatase-related protein type 3；
12) searching number is P50748, and gene annotation is Kinetochore-associated protein 1；
13) searching number is Q8IVL1, and gene annotation is Neuron navigator 2；
14) searching number is P60953, and gene annotation is Cell division control protein 42homolog；
15) searching number is P02808, gene annotation Statherin；
16) searching number is Q9UKK3, and gene annotation is Poly [ADP-ribose] polymerase 4；
17) searching number is P78371, and gene annotation is T-complex protein 1subunit beta；
18) searching number is O00602, gene annotation Ficolin-1；
19) searching number is P22392, and gene annotation is Nucleoside diphosphate kinase B；
20) searching number is Q9BRZ2, and gene annotation is E3ubiquitin-protein ligase TRIM56；
21) searching number is O75015, and gene annotation is Low affinity immunoglobulin gamma Fc region receptor III-B；
22) searching number is Q15493, gene annotation Regucalcin；
23) searching number is P01703, and gene annotation is Ig lambda chain V-I region NEWM；
24) searching number is P16401, and gene annotation is Histone H1.5；
25) searching number is P80303, gene annotation Nucleobindin-2；
26) searching number is P32243, and gene annotation is Homeobox protein OTX2；
27) searching number is Q16378, and gene annotation is Proline-rich protein 4；
28) searching number is Q7Z5M8, and gene annotation is Abhydrolase domain-containing protein 12B；
29) searching number is P58499, and gene annotation is Protein FAM3B；
30) searching number is P42765, and gene annotation is 3-ketoacyl-CoA thiolase, mitochondrial；
31) searching number is Q9P0M2, and gene annotation is A-kinase anchor protein 7isoform gamma；
32) searching number is P22626, and gene annotation is Heterogeneous nuclear ribonucleoproteins A2/ B1；
33) searching number is Q9UNU6, and gene annotation is 7-alpha-hydroxycholest-4-en-3-one 12-alpha- hydroxylase；
34) searching number is P20929, gene annotation Nebulin；
35) searching number is P04259, gene annotation Keratin, type II cytoskeletal 6B；
36) searching number is P04438, and gene annotation is Ig heavy chain V-II region SESS；
37) searching number is O43852, gene annotation Calumenin；
38) searching number is Q9UHY1, and gene annotation is Nuclear receptor-binding protein；
39) searching number is Q12841, and gene annotation is Follistatin-related protein 1；
40) searching number is Q9UPT6, and gene annotation is C-Jun-amino-terminal kinase-interacting protein 3；
41) searching number is Q06828, gene annotation Fibromodulin；
42) searching number is Q9H4M9, and gene annotation is EH domain-containing protein 1；
43) searching number is Q04917 14-3-3, and gene annotation is protein eta；
44) searching number is Q9HCU9, and gene annotation is Breast cancer metastasis-suppressor 1；
45) searching number is P05089, gene annotation Arginase-1；
46) searching number is Q96RS0, and gene annotation is Trimethylguanosine synthase；
47) searching number is Q8IWF6, and gene annotation is Protein DENND6A；
48) searching number is Q92521, and gene annotation is GPI mannosyltransferase 3；
49) searching number is P33241, and gene annotation is Lymphocyte-specific protein 1；
50) searching number is Q96FZ7, and gene annotation is Charged multivesicular body protein 6；
51) searching number is Q9ULZ3, and gene annotation is Apoptosis-associated speck-like protein containing a CARD；
52) searching number is P25787, and gene annotation is Proteasome subunit alpha type-2；
53) searching number is O60361, and gene annotation is Putative nucleoside diphosphate kinase；
54) searching number is Q8TAX7, gene annotation Mucin-7；
55) searching number is Q92688, and gene annotation is Acidic leucine-rich nuclear phosphoprotein 32 family member B；
56) searching number is P40394, and gene annotation is Alcohol dehydrogenase class 4mu/sigma chain；
57) searching number is Q14289, and gene annotation is Protein-tyrosine kinase 2-beta；
58) searching number is P55008, and gene annotation is Allograft inflammatory factor 1；
59) searching number is P08571, and gene annotation is Monocyte differentiation antigen CD14；
60) searching number is P21439, and gene annotation is Multidrug resistance protein3；
61) searching number is O95777U6, and gene annotation is snRNA-associated Sm-like protein LSm8；
62) searching number is Q96DA0, and gene annotation is Zymogen granule protein 16homolog B；
63) searching number is Q16651, gene annotation Prostasin；
64) searching number is Q9Y4Y9, and gene annotation is U6snRNA-associated Sm-like protein LSm5；
65) searching number is P26583, and gene annotation is High mobility group protein B2；
66) searching number is Q9H008, and gene annotation is Phospholysine phosphohistidine inorganic pyrophosphate phosphatase；
67) searching number is P01742, and gene annotation is Ig heavy chain V-I region EU；
68) searching number is Q15631, gene annotation Translin；
69) searching number is Q9HC38, and gene annotation is Glyoxalase domain-containing protein 4；
70) searching number is Q9Y376, and gene annotation is Calcium-binding protein 39；
71) searching number is Q96DR5, and gene annotation is BPI fold-containing family A member 2；
72) searching number is P07477Trypsin-1；
73) searching number is Q96C19, and gene annotation is EF-hand domain-containing protein D2；
74) searching number is P16870, and gene annotation is Carboxypeptidase E；
75) searching number is Q8NI35, and gene annotation is InaD-like protein；
76) searching number is P05362, and gene annotation is Intercellular adhesion molecule 1；
77) searching number is P15374, and gene annotation is Ubiquitin carboxyl-terminal hydrolase isozyme L3；
78) searching number is Q6ZNW5, and gene annotation is GDP-D-glucose phosphorylase 1；
79) searching number is Q15370, and gene annotation is Transcription elongation factor B polypeptide 2；
80) searching number is P0CG05, and gene annotation is Ig lambda-2chain C regions；
81) searching number is P30740, and gene annotation is Leukocyte elastase inhibitor；
82) searching number is Q7Z6J4, gene annotation FYVE, RhoGEF and PH domain-containing protein 2；
83) searching number is Q9H7L9, and gene annotation is Sin3histone deacetylase corepressor complex component SDS3；
84) searching number is P00533, and gene annotation is Epidermal growth factor receptor；
85) searching number is P30046, and gene annotation is D-dopachrome decarboxylase；
86) searching number is Q96BQ1, and gene annotation is Protein FAM3D；
87) searching number is Q9NP98, gene annotation Myozenin-1；
88) searching number is P31146, gene annotation Coronin-1A；
89) searching number is O75503, and gene annotation is Ceroid-lipofuscinosis neuronal protein 5；
90) searching number is P63279, and gene annotation is SUMO-conjugating enzyme UBC9；
91) searching number is Q8IUE6, and gene annotation is Histone H2A type 2-B；
92) searching number is Q9NZ53, and gene annotation is Podocalyxin-like protein 2；
93) searching number is Q8NF91, gene annotation Nesprin-1；
94) searching number is Q9BS40, gene annotation Latexin；
95) searching number is P09382, gene annotation Galectin-1；
96) searching number is Q9BRF8, and gene annotation is Serine/threonine-protein phosphatase CPPED1；
97) searching number is P08579, and gene annotation is U2small nuclear ribonucleoprotein B "；
98) searching number is P49720, and gene annotation is Proteasome subunit beta type-3；
99) searching number is P52566, and gene annotation is Rho GDP-dissociation inhibitor 2；
100) searching number is Q8NC60, and gene annotation is Nitric oxide-associated protein 1；
101) searching number is P10909, gene annotation Clusterin；
102) searching number is P01712, and gene annotation is glambda chain V-II region WIN；
103) searching number is P23280, and gene annotation is Carbonic anhydrase 6；
104) searching number is Q8IWQ3, and gene annotation is Serine/threonine-protein kinase BRSK2；
105) searching number is Q86VR7, and gene annotation is V-set and immunoglobulin domain-containing protein 10-like；
106) searching number is Q99439, gene annotation Calponin-2；
107) searching number is Q9GZZ8, and gene annotation is Extracellular glycoprotein lacritin；
108) searching number is O43707, gene annotation Alpha-actinin-4；
109) searching number is P62253, and gene annotation is Ubiquitin-conjugating enzyme E2G1；
110) searching number is Q13421, gene annotation Mesothelin；
111) searching number is P35237, and gene annotation is Serpin B6；
112) searching number is P14550, and gene annotation is Alcohol dehydrogenase [NADP (+)]；
113) searching number is P06737, and gene annotation is Glycogen phosphorylase, liver form；
114) searching number is O00584, and gene annotation is Ribonuclease T2；
115) searching number is P62993, and gene annotation is Growth factor receptor-bound protein 2；
116) searching number is P01707, and gene annotation is Ig lambda chain V-II region TRO；
117) searching number is P01036, gene annotation Cystatin-S；
118) searching number is P35908, gene annotation Keratin, type II cytoskeletal 2epidermal；
119) searching number is O95684, and gene annotation is FGFR1oncogene partner；
120) searching number is O75891, and gene annotation is Cytosolic 10-formyltetrahydrofolate dehydrogenase；
121) searching number is Q86VD1, and gene annotation is MORC family CW-type zinc finger protein 1；
122) searching number is Q9Y490, gene annotation Talin-1；
123) searching number is P05154, and gene annotation is Plasma serine protease inhibitor；
124) searching number is O15143, and gene annotation is Actin-related protein 2/3complex subunit 1B；
125) searching number is O75563, and gene annotation is Src kinase-associated phosphoprotein 2；
126) searching number is A0M8Q6, and gene annotation is Ig lambda-7chain C region；
127) searching number is Q6JBY9, and gene annotation is CapZ-interacting protein.
CN201711241298.5A 2017-11-30 2017-11-30 The method of liver cancer syndrome of spleen deficiency biological marker analyte detection based on sialoprotein matter group Pending CN108152357A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112345607A (en) * 2020-10-12 2021-02-09 苏州健雄职业技术学院 Triazophos biosensor based on two-dimensional nano material
CN113407797A (en) * 2021-08-18 2021-09-17 成都千嘉科技有限公司 Data mining method for gas stealing behavior by utilizing fractal calculation
CN113495142A (en) * 2020-03-20 2021-10-12 深圳市第二人民医院(深圳市转化医学研究院) Gastric cancer spleen deficiency syndrome sialoprotein marker and detection method and application thereof

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113495142A (en) * 2020-03-20 2021-10-12 深圳市第二人民医院(深圳市转化医学研究院) Gastric cancer spleen deficiency syndrome sialoprotein marker and detection method and application thereof
CN112345607A (en) * 2020-10-12 2021-02-09 苏州健雄职业技术学院 Triazophos biosensor based on two-dimensional nano material
CN113407797A (en) * 2021-08-18 2021-09-17 成都千嘉科技有限公司 Data mining method for gas stealing behavior by utilizing fractal calculation
CN113407797B (en) * 2021-08-18 2021-10-29 成都千嘉科技有限公司 Data mining method for gas stealing behavior by utilizing fractal calculation

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