CN108107219A

CN108107219A - The method of liver cancer syndrome of deficiency of kidney yin biological marker analyte detection based on sialoprotein matter group

Info

Publication number: CN108107219A
Application number: CN201711241300.9A
Authority: CN
Inventors: 吴正治; 孙珂焕; 丁峰; 黄飞娟; 曹美群; 范大华; 姚永超
Original assignee: Shenzhen Institute of Gerontology
Current assignee: Shenzhen Institute of Gerontology
Priority date: 2017-11-30
Filing date: 2017-11-30
Publication date: 2018-06-01

Abstract

The invention discloses the methods of the liver cancer syndrome of deficiency of kidney yin 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 label in liver cancer syndrome of deficiency of kidney yin, 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 deficiency of kidney yin, postoperative recurrence, transfer and Observation On The Prognosis.

Description

The method of liver cancer syndrome of deficiency of kidney yin biological marker analyte detection based on sialoprotein matter group

Technical field

The present invention relates to a kind of biological marker object detecting methods, are specifically a kind of liver cancer kidney based on sialoprotein matter group The method of yin deficiency syndrome 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, all protein of tissue or even cell, 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 be subject to 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 Marking protein finds out the protein of the next body differential expression of different conditions, so as to be material base, the morning of disease of 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 substantial amounts of hormone, protein, enzyme, antibody, complement, thin The blood constituents such as intracellular cytokine and various microorganisms, these blood constituents pass through along the passive cross-cell membrane transhipment or inverse of concentration gradient The approach such as the active transport of concentration gradient are secreted among 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 Relation, 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 for the 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 having found 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, it is necessary to 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.

The content of the invention

It is an object of the invention to provide a kind of safe and reliable, high efficiency liver cancer kidneys based on sialoprotein matter group The method of yin deficiency syndrome 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 deficiency of kidney yin 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 relation 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；The BSA digested using 400 μ g is carried out 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 separated, 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 nephrasthenia syndrome and Normal group Fruit shows：Identify 1297 albumen；Compared with Normal group, 1.5 times or more differential expressions being identified in liver cancer nephrasthenia syndrome 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 Q96JC1, and gene annotation is Vam6/Vps39-like protein；

2) searching number is O00755, and gene annotation is Protein Wnt-7a；

3) searching number is P06702, and gene annotation is Protein S100-A9；

4) searching number is Q6ZP82, and gene annotation is Coiled-coil domain-containing protein 141；

5) searching number is P05109, and gene annotation is Protein S100-A8；

6) searching number is Q8NDA2, gene annotation Hemicentin-2；

7) searching number is Q7Z589, and gene annotation is Protein EMSY；

8) searching number is Q6UW32, and gene annotation is Insulin growth factor-like family member 1；

9) searching number is Q6ZT21, and gene annotation is Transmembrane protein with metallophosphoesterase domain；

10) searching number is P51159, and gene annotation is Ras-related protein Rab-27A；

11) searching number be Q14573, gene annotation be Inositol Isosorbide-5-Nitraes, 5-trisP29034hosphate receptor type 3；

12) searching number is P0CG06, and gene annotation is Ig lambda-3chain C regions；

13) searching number is P35354, and gene annotation is Prostaglandin G/H synthase 2；

14) searching number is P05164, gene annotation Myeloperoxidase；

15) searching number is Q14508, and gene annotation is WAP four-disulfide core domain protein 2；

16) searching number is P83593, and gene annotation is g kappa chain V-IV region STH (Fragment；

17) searching number is O75629, and gene annotation is Protein CREG1；

18) searching number is Q92565, and gene annotation is Rap guanine nucleotide exchange factor 5；

19) searching number is P02795, gene annotation Metallothionein-2；

20) searching number is Q02218, and gene annotation is 2-oxoglutarate dehydrogenase, mitochondrial；

21) searching number is P55145, and gene annotation is Mesencephalic astrocyte-derived neurotrophic factor；

22) searching number is P02792, and gene annotation is Ferritin light chain；

23) searching number is P50552, and gene annotation is Vasodilator-stimulated phosphoprotein；

24) searching number is P02771, gene annotation Alpha-fetoprotein；

25) searching number is Q16623, gene annotation Syntaxin-1A；

26) searching number is P22532, and gene annotation is Small proline-rich protein 2D；

27) searching number is P00390, and gene annotation is Glutathione reductase, mitochondrial；

28) searching number is P34972, and gene annotation is Cannabinoid receptor 2；

29) searching number is Q96JB2, and gene annotation is Conserved oligomeric Golgi complex subunit 3；

30) searching number is Q9HD89, gene annotation Resistin；

31) searching number is P03973, gene annotation Antileukoproteinase；

32) searching number is P04179, and gene annotation is Superoxide dismutase [Mn], mitochondrial；

33) searching number is P01040, gene annotation Cystatin-A；

34) searching number is Q9UL25, and gene annotation is Ras-related protein Rab-21；

35) searching number is P28749, and gene annotation is Retinoblastoma-like protein 1；

36) searching number is P02743, and gene annotation is Serum amyloid P-component；

37) searching number is P58062, and gene annotation is Serine protease inhibitor Kazal-type 7；

38) searching number is P00736, and gene annotation is Complement C1r subcomponent；

39) searching number is Q96T37, and gene annotation is Putative RNA-binding protein 15；

40) searching number is P01770, and gene annotation is Ig heavy chain V-III region NIE；

41) searching number is P08246, and gene annotation is Neutrophil elastase；

42) searching number is P20160, gene annotation Azurocidin；

43) searching number is Q7L0X2, and gene annotation is Glutamate-rich protein 6；

44) searching number is P10643, and gene annotation is Complement component C7；

45) searching number is P04181, and gene annotation is Ornithine aminotransferase, mitochondrial；

46) searching number is Q9H7L9, and gene annotation is Sin3histone deacetylase corepressor complex component SDS3；

47) searching number is P29034, and gene annotation is Protein S100-A2；

48) searching number is O75038, and gene annotation is 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase eta-2；

49) searching number is Q8NCM8, gene annotation is Cytoplasmic dynein 2heavy chain 1；

50) searching number is Q9Y6V0, and gene annotation is Protein piccolo；

51) searching number is P01743, and gene annotation is Ig heavy chain V-I region HG3；

52) searching number is P04632, and gene annotation is Calpain small subunit 1；

53) searching number is Q5T5U3, and gene annotation is Rho GTPase-activating protein 21；

54) searching number is P14174, and gene annotation is Macrophage migration inhibitory factor；

55) searching number is O43653, and gene annotation is Prostate stem cell antigen；

56) searching number is P01612, and gene annotation is Ig kappa chain V-I region Mev；

57) searching number is Q14210, and gene annotation is Lymphocyte antigen 6D；

58) searching number is P12883, gene annotation Myosin-7；

59) searching number is P80188, and gene annotation is Neutrophil gelatinase-associated lipocalin；

60) searching number is P31997, and gene annotation is Carcinoembryonic antigen-related cell adhesion molecule 8；

61) searching number is Q10588, and gene annotation is ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 2；

62) searching number is P02763, and gene annotation is Alpha-1-acid glycoprotein 1；

63) searching number is Q9UBW5, and gene annotation is Bridging integrator 2；

64) searching number is P02788, gene annotation Lactotransferrin；

65) searching number is Q66K66, and gene annotation is Transmembrane protein 198；

66) searching number is P08236, gene annotation Beta-glucuronidase；

67) searching number is O60635, gene annotation Tetraspanin-1；

68) searching number is P14780, and gene annotation is Matrix metalloproteinase-9；

69) searching number is P13473, and gene annotation is Lysosome-associated membrane glycoprotein 2；

70) searching number is P13010, and gene annotation is X-ray repair cross-complementing protein 5；

71) searching number is P04003, and gene annotation is C4b-binding protein alpha chain；

72) searching number is P01624, and gene annotation is Ig kappa chain V-III region POM；

73) searching number is P06748, gene annotation Nucleophosmin；

74) searching number is O75578, and gene annotation is Integrin alpha-10；

75) searching number is P59665, and gene annotation is Neutrophil defensin 1；

76) searching number is P02741, and gene annotation is C-reactive protein；

77) searching number is Q8N6Q3, gene annotation CD177antigen；

78) searching number is P46939, gene annotation Utrophin；

79) searching number is Q9P1Z9, and gene annotation is Coiled-coil domain-containing protein 180；

80) searching number is Q9NQ48, and gene annotation is Leucine zipper transcription factor-like protein 1；

81) searching number is P00738, gene annotation Haptoglobin；

82) searching number is P55000, and gene annotation is Secreted Ly-6/uPAR-related protein 1；

83) searching number is P01771, and gene annotation is Ig heavy chain V-III region HIL；

84) searching number is P49913, and gene annotation is Cathelicidin antimicrobial peptide；

85) searching number is Q9UFN0, and gene annotation is Protein NipSnap homolog 3A；

86) searching number is P07602, gene annotation Prosaposin；

87) searching number is Q96AT9, and gene annotation is Ribulose-phosphate 3-epimerase；

88) searching number is Q96L46, and gene annotation is Calpain small subunit 2；

89) searching number is P02760, and gene annotation is Protein AMBP；

90) searching number is P01773, and gene annotation is Ig heavy chain V-III region BUR；

91) searching number is P69905, and gene annotation is Hemoglobin subunit alpha；

92) searching number is P49221, and gene annotation is Protein-glutamine gamma- glutamyltransferase 4；

93) searching number is P36222, and gene annotation is Chitinase-3-like protein 1；

94) searching number is P01764, and gene annotation is Ig heavy chain V-III region 23；

95) searching number is P19652, and gene annotation is Alpha-1-acid glycoprotein 2；

96) searching number is P28799, gene annotation Granulins；

97) searching number is P00505, and gene annotation is Aspartate aminotransferase, mitochondrial；

98) searching number is P00441, and gene annotation is Superoxide dismutase [Cu-Zn]；

99) searching number is P01598, and gene annotation is Ig kappa chain V-I region EU；

100) searching number is Q12913, and gene annotation is Receptor-type tyrosine-protein phosphatase eta；

101) searching number is P20933, and gene annotation is N (4)-(beta-N-acetylglucosaminyl)-L- asparaginase；

102) searching number is P17900, and gene annotation is Ganglioside GM2activator；

103) searching number is Q8WWY7, WAP, and gene annotation is four-disulfide core domain protein 12；

104) searching number is P22894, and gene annotation is Neutrophil collagenase；

105) searching number is O95445, and gene annotation is Apolipoprotein M；

106) searching number is P04839, and gene annotation is Cytochrome b-245heavy chain；

107) searching number is P02753, and gene annotation is Retinol-binding protein 4；

108) searching number is Q5JS37, and gene annotation is NHL repeat-containing protein 3；

109) searching number is A6NKB5, and gene annotation is Pecanex-like protein 2；

110) searching number is P15090, and gene annotation is Fatty acid-binding protein, adipocyte；

111) searching number is Q9GZP4, and gene annotation is PITH domain-containing protein 1；

112) searching number is P01623, and gene annotation is Ig kappa chain V-III region WOL；

113) searching number is P02794, and gene annotation is Ferritin heavy chain；

114) searching number is P61457, gene annotation Pterin-4-alpha-carbinolamine dehydratase；

115) searching number is P10153, and gene annotation is Non-secretory ribonuclease；

116) searching number is P42768, and gene annotation is Wiskott-Aldrich syndrome protein；

117) searching number is O75594, and gene annotation is Peptidoglycan recognition protein 1；

118) searching number is Q9H244, and gene annotation is P2Y purinoceptor 12；

119) searching number is P00387, and gene annotation is NADH-cytochrome b5reductase 3；

120) searching number is P63208, and gene annotation is S-phase kinase-associated protein 1

121) searching number is P02748, and gene annotation is Complement component C9；

122) searching number is P01782, and gene annotation is Ig heavy chain V-III region DOB；

123) searching number is P68871, and gene annotation is Hemoglobin subunit beta；

124) searching number is Q04837, and gene annotation is Single-stranded DNA-binding protein, mitochondrial；

125) searching number is Q86VH2, and gene annotation is Kinesin-like protein KIF27；

126) searching number is Q9ULD9, and gene annotation is Zinc finger protein 608；

127) searching number is Q7RTS6, gene annotation Otopetrin-2；

128) searching number is P01834, and gene annotation is Ig kappa chain C region；

129) searching number is Q9NQ38, and gene annotation is Serine protease inhibitor Kazal-type 5；

130) searching number is O60701, and gene annotation is UDP-glucose 6-dehydrogenase；

131) searching number is P50995, and gene annotation is Annexin A11；

132) searching number is Q9H0W9, and gene annotation is Ester hydrolase C11orf54；

133) searching number is Q8IUX8, and gene annotation is Epidermal growth factor-like protein 6；

134) searching number is P10155, and gene annotation is 60kDa SS-A/Ro ribonucleoprotein；

135) searching number is P06870, gene annotation Kallikrein-1；

136) searching number is P01877, and gene annotation is Ig alpha-2chain C region；

137) searching number is Q9BPV8, and gene annotation is P2Y purinoceptor 13；

138) searching number is P13987, gene annotation CD59glycoprotein；

139) searching number is Q9BRT3, and gene annotation is Migration and invasion enhancer 1；

140) searching number is P13688, and gene annotation is Carcinoembryonic antigen-related cell adhesion molecule 1；

141) searching number is P09525, and gene annotation is Annexin A4；

142) searching number is O15400, gene annotation Syntaxin-7；

143) searching number is P31949, and gene annotation is Protein S100-A11；

144) searching number is Q6P4A8, and gene annotation is Phospholipase B-like 1；

145) searching number is Q96RM1, and gene annotation is Small proline-rich protein 2F；

146) searching number is O43866, gene annotation CD5antigen-like；

147) searching number is P01617, and gene annotation is Ig kappa chain V-II region TEW；

148) searching number is P05155, and gene annotation is Plasma protease C1inhibitor；

149) searching number is P04434, and gene annotation is Ig kappa chain V-III region VH (Fragment)；

150) searching number is P48637, and gene annotation is Glutathione synthetase；

151) searching number is P98088, gene annotation Mucin-5AC；

152) searching number is O95479, and gene annotation is GDH/6PGL endoplasmic bifunctional protein；

153) searching number is P35241, gene annotation Radixin；

154) searching number is P80419, and gene annotation is Ig heavy chain V-III region GAR；

155) searching number is P25774, and gene annotation is Cathepsin S；

156) searching number is P08603, and gene annotation is Complement factor H；

157) searching number is Q9UGM3, gene annotation is Deleted in malignant brain tumors 1protein；

158) searching number is Q16881, and gene annotation is Thioredoxin reductase 1, cytoplasmic；

159) searching number is P01763, and gene annotation is Ig heavy chain V-III region WEA；

160) searching number is P19957, gene annotation Elafin；

161) searching number is Q6UXH1, and gene annotation is Cysteine-rich with EGF-like domain protein 2；

162) searching number is P01766, and gene annotation is Ig heavy chain V-III region BRO；

163) searching number is O14732, and gene annotation is Inositol monophosphatase 2；

164) searching number is P05107, and gene annotation is Integrin beta-2；

165) searching number is O43451, gene annotation Maltase-glucoamylase, intestinal；

166) searching number is O43768, gene annotation Alpha-endosulfine；

167) searching number is P16219, and gene annotation is Short-chain specific acyl-CoA dehydrogenase, mitochondrial；

168) searching number is P02790, gene annotation Hemopexin；

169) searching number is Q00839, and gene annotation is Heterogeneous nuclear ribonucleoprotein U；

170) searching number is P31151, and gene annotation is Protein S100-A7；

171) searching number is P06331, and gene annotation is Ig heavy chain V-II region ARH-77；

172) searching number is P42126, and gene annotation is Enoyl-CoA delta isomerase 1, mitochondrial；

173) searching number is P01857, and gene annotation is Ig gamma-1chain C region；

174) searching number is P01616, and gene annotation is Ig kappa chain V-II region MIL；

175) searching number is O75635, and gene annotation is Serpin B7；

176) searching number is P15328, and gene annotation is Folate receptor alpha；

177) searching number is P08758, and gene annotation is Annexin A5；

178) searching number is Q8TED4, and gene annotation is Sugar phosphate exchanger 2；

179) searching number is O15031, gene annotation Plexin-B2；

180) searching number is P06731, and gene annotation is Carcinoembryonic antigen-related cell adhesion molecule 5；

181) searching number is P01861, and gene annotation is Ig gamma-4chain C region；

182) searching number is P23083, and gene annotation is Ig heavy chain V-I region V35；

183) searching number is P01033, and gene annotation is Metalloproteinase inhibitor 1；

184) searching number is Q99828, and gene annotation is Calcium and integrin-binding protein 1；

185) searching number is P01776, and gene annotation is Ig heavy chain V-III region WAS；

186) searching number is Q86SG5, and gene annotation is Protein S100-A7A；

187) searching number is Q9Y608, and gene annotation is Leucine-rich repeat flightless- interacting protein 2

188) searching number is P01023, gene annotation Alpha-2-macroglobulin；

189) searching number is Q969T9, and gene annotation is WW domain-binding protein 2；

190) searching number is P01762, and gene annotation is Ig heavy chain V-III region TRO；

191) searching number is P27695, and gene annotation is DNA- (apurinic or apyrimidinic site) lyase；

192) searching number is P01767, and gene annotation is Ig heavy chain V-III region BUT；

193) searching number is P01605, and gene annotation is Ig kappa chain V-I region Lay；

194) searching number is Q6PCB0, and gene annotation is von Willebrand factor A domain- containing protein 1；

195) searching number is Q9UL46, and gene annotation is Proteasome activator complex subunit 2；

196) searching number is P01876, and gene annotation is Ig alpha-1chain C region；

197) searching number is P01781, and gene annotation is Ig heavy chain V-III region GAL；

198) searching number is P00734, gene annotation Prothrombin；

199) searching number is Q99584, and gene annotation is Protein S100-A13；

200) searching number is P10644, and gene annotation is cAMP-dependent protein kinase type I- alpha regulatory subunit；

201) searching number is P54108, and gene annotation is Cysteine-rich secretory protein 3；

202) searching number is P02042, and gene annotation is Hemoglobin subunit delta；

203) searching number is P01859, and gene annotation is Ig gamma-2chain C region；

204) searching number is P01603, and gene annotation is Ig kappa chain V-I region Ka；

Down-regulation protein is as follows：

1) searching number is Q2TAA2, and gene annotation is Isoamyl acetate-hydrolyzing esterase 1homolog；

2) searching number is O60814, and gene annotation is Histone H2B type 1-K；

3) searching number is Q06828, gene annotation Fibromodulin；

4) searching number is P02808, gene annotation Statherin；

5) searching number is F5H4A9, and gene annotation is Uncharacterized membrane protein C3orf80；

6) searching number is Q8IVL1, and gene annotation is Neuron navigator 2；

7) searching number is P22392, and gene annotation is Nucleoside diphosphate kinase B；

8) searching number is P80303, gene annotation Nucleobindin-2；

9) searching number is Q96A08, and gene annotation is Histone H2B type 1-A；

10) searching number is Q9BYB0, and gene annotation is SH3and multiple ankyrin repeat domains protein 3；

11) searching number is P04745, and gene annotation is Alpha-amylase 1；

12) searching number is Q8IUE6, and gene annotation is Histone H2A type 2-B；

13) searching number is Q9H2E6, gene annotation Semaphorin-6A；

14) searching number is Q2NL68, and gene annotation is Proline and serine-rich protein 3；

15) searching number is P01602, and gene annotation is Ig heavy chain V-I region 5 (Fragment)；

16) searching number is P35754, gene annotation Glutaredoxin-1；

17) searching number is Q5SNV9, and gene annotation is Uncharacterized protein C1orf167；

18) searching number is Q7Z5M8, gene annotation is Abhydrolase domain-containing protein 12B；

19) searching number is Q96DR5, and gene annotation is BPI fold-containing family A member 2；

20) searching number is Q15181, and gene annotation is Inorganic pyrophosphatase；

21) searching number is Q99460, and gene annotation is 26S proteasome non-ATPase regulatory subunit 1；

22) searching number is Q9BPY8, and gene annotation is Homeodomain-only protein；

23) searching number is Q9GZZ8, and gene annotation is Extracellular glycoprotein lacritin；

24) searching number is P32243, and gene annotation is Homeobox protein OTX2；

25) searching number is O95881, and gene annotation is Thioredoxin domain-containing protein 12；

26) searching number is Q9H299, and gene annotation is SH3domain-binding glutamic acid-rich- like protein 3；

27) searching number is Q92520, and gene annotation is Protein FAM3C；

28) searching number is P23280, and gene annotation is Carbonic anhydrase 6；

29) searching number is Q9BRK5, and gene annotation is 45kDa calcium-binding protein；

30) searching number is P22626, and gene annotation is Heterogeneous nuclear ribonucleoproteins A2/B1；

31) searching number is Q16378, and gene annotation is Proline-rich protein 4；

32) searching number is Q14118, gene annotation Dystroglycan；

33) searching number is P0C0S5, and gene annotation is Histone H2A.Z；

34) searching number is P16401, and gene annotation is Histone H1.5；

35) searching number is P02652, and gene annotation is Apolipoprotein A-II；

36) searching number is Q9BRZ2, and gene annotation is E3ubiquitin-protein ligase TRIM56；

37) searching number is Q13185, and gene annotation is Chromobox protein homolog 3；

38) searching number is P16870, and gene annotation is Carboxypeptidase E；

39) searching number is Q7Z591, and gene annotation is AT-hook-containing transcription factor；

40) searching number is O15050, and gene annotation is TPR and ankyrin repeat-containing protein 1；

41) searching number is Q9UKK3, and gene annotation is Poly [ADP-ribose] polymerase 4；

42) searching number is P08571, and gene annotation is Monocyte differentiation antigen CD14；

43) searching number is P28838, and gene annotation is Cytosol aminopeptidase；

44) searching number is Q8N4F0, and gene annotation is BPI fold-containing family B member 2；

45) searching number is O43852, gene annotation Calumenin；

46) searching number is Q13127, and gene annotation is RE1-silencing transcription factor；

47) searching number is P50748, and gene annotation is Kinetochore-associated protein 1；

48) searching number is P48735, and gene annotation is Isocitrate dehydrogenase [NADP], mitochondrial；

49) searching number is Q9HAJ7, and gene annotation is Histone deacetylase complex subunit SAP30L；

50) searching number is Q6ZUA9, and gene annotation is Maestro heat-like repeat family member 5；

51) searching number is Q96FZ7, and gene annotation is Charged multivesicular body protein 6；

52) searching number is O75695, and gene annotation is Protein XRP2；

53) searching number is Q9H4M9, and gene annotation is EH domain-containing protein 1；

54) searching number is P01036, gene annotation Cystatin-S；

55) searching number is Q99592, and gene annotation is Zinc finger and BTB domain-containing protein 18；

56) searching number is P04792, and gene annotation is Heat shock protein beta-1；

57) searching number is Q04917, and gene annotation is 14-3-3protein eta；

58) searching number is P58499, and gene annotation is Protein FAM3B；

59) searching number is P30085, and gene annotation is UMP-CMP kinase；

60) searching number is Q9P0M2, and gene annotation is A-kinase anchor protein 7isoform gamma；

61) searching number is P30405, and gene annotation is Peptidyl-prolyl cis-trans isomerase F, mitochondrial；

62) searching number is P02647, and gene annotation is Apolipoprotein A-I；

63) searching number is P15374, and gene annotation is Ubiquitin carboxyl-terminal hydrolase isozyme L3；

64) searching number is Q96DA0, and gene annotation is Zymogen granule protein 16homolog B；

65) searching number is P62805, and gene annotation is Histone H4；

66) searching number is P55957, and gene annotation is BH3-interacting domain death agonist；

67) searching number is Q05315, gene annotation Galectin-10；

68) searching number is Q14289, and gene annotation is Protein-tyrosine kinase 2-beta；

69) searching number is P15515, gene annotation Histatin-1；

70) searching number is Q9H2U2, and gene annotation is Inorganic pyrophosphatase 2, mitochondrial；

71) searching number is P28325, gene annotation Cystatin-D；

72) searching number is P63313, and gene annotation is Thymosin beta-10；

73) searching number is P06737, and gene annotation is Glycogen phosphorylase, liver form；

74) searching number is P30044, gene annotation Peroxiredoxin-5, mitochondrial；

75) searching number is O95777, and gene annotation is U6snRNA-associated Sm-like protein LSm8；

76) searching number is P40925, and gene annotation is Malate dehydrogenase, cytoplasmic；

77) searching number is O75015, and gene annotation is Low affinity immunoglobulin gamma Fc region receptor III-B；

78) searching number is P68032, gene annotation Actin, alpha cardiac muscle 1；

79) searching number is P15289, and gene annotation is Arylsulfatase A；

80) searching number is O00602, gene annotation Ficolin-1；

81) searching number is P40121, and gene annotation is Macrophage-capping protein；

82) searching number is P04406, gene annotation Glyceraldehyde-3-phosphate dehydrogenase；

83) searching number is Q9UBC9, and gene annotation is Small proline-rich protein 3；

84) searching number is Q8NBJ4, and gene annotation is Golgi membrane protein 1；

85) searching number is Q96FV2, gene annotation Secernin-2；

86) searching number is Q96BQ1, and gene annotation is Protein FAM3D；

87) searching number is Q9NRS6, and gene annotation is Sorting nexin-15；

88) searching number is P05089, gene annotation Arginase-1；

89) searching number is Q6BCY4, and gene annotation is NADH-cytochrome b5reductase 2；

90) searching number is Q9Y5Z4, and gene annotation is Heme-binding protein 2；

91) searching number is P11216, and gene annotation is Glycogen phosphorylase, brain form；

92) searching number is P60709, gene annotation Actin, cytoplasmic 1；

93) searching number is P30041, gene annotation Peroxiredoxin-6；

94) searching number is P20962, gene annotation Parathymosin；

95) searching number is Q9H8J5, and gene annotation is MANSC domain-containing protein 1；

96) searching number is P68431, and gene annotation is Histone H3.1；

97) searching number is P23381, and gene annotation is Tryptophan--tRNA ligase, cytoplasmic；

98) searching number is P07737, gene annotation Profilin-1；

99) searching number is P01712, and gene annotation is Ig lambda chain V-II region WIN；

100) searching number is P46108, and gene annotation is Adapter molecule crk；

101) searching number is O00584, and gene annotation is Ribonuclease T2；

102) searching number is Q6NUM9, and gene annotation is All-trans-retinol 13,14-reductase；

103) searching number is P19827, and gene annotation is Inter-alpha-trypsin inhibitor heavy chain H1；

104) searching number is Q02413, gene annotation Desmoglein-1；

105) searching number is O60361, and gene annotation is Putative nucleoside diphosphate kinase；

106) searching number is O95861, gene annotation 3'(2'), 5'-bisphosphate nucleotidase 1；

107) searching number is P55058, and gene annotation is Phospholipid transfer protein；

108) searching number is Q9UHY7, and gene annotation is Enolase-phosphatase E1；

109) searching number is Q13043, and gene annotation is Serine/threonine-protein kinase 4；

110) searching number is P61956, and gene annotation is Small ubiquitin-related modifier 2；

111) searching number is Q8TAX7, gene annotation Mucin-7；

112) searching number is P0C0L4, and gene annotation is Complement C4-A；

113) searching number is Q96C23, and gene annotation is Aldose 1-epimerase；

114) searching number is P11233, and gene annotation is Ras-related protein Ral-A；

115) searching number is P22314, and gene annotation is Ubiquitin-like modifier-activating enzyme 1；

116) searching number is Q15493, gene annotation Regucalcin；

117) searching number is P40394, and gene annotation is Alcohol dehydrogenase class 4mu/sigma chain；

118) searching number is Q9UHA7, gene annotation Interleukin-36alpha；

119) searching number is Q9UBX7, gene annotation Kallikrein-11；

120) searching number is P23528, gene annotation Cofilin-1；

121) searching number is P00338, and gene annotation is L-lactate dehydrogenase A chain；

122) searching number is Q8NI35, and gene annotation is InaD-like protein；

123) searching number is Q15121, and gene annotation is Astrocytic phosphoprotein PEA-15；

124) searching number is P52272, and gene annotation is Heterogeneous nuclear ribonucleoprotein M；

125) searching number is P01037, gene annotation Cystatin-SN；

126) searching number is O75367, and gene annotation is Core histone macro-H2A.1；

127) searching number is Q8IWF6, and gene annotation is Protein DENND6A；

128) searching number is P61960, and gene annotation is Ubiquitin-fold modifier 1；

129) searching number is Q9Y3C8, and gene annotation is Ubiquitin-fold modifier-conjugating enzyme 1；

130) searching number is P30043, and gene annotation is Flavin reductase (NADPH)；

131) searching number is P25787, and gene annotation is Proteasome subunit alpha type-2；

132) searching number is P09871, and gene annotation is Complement C1s subcomponent；

133) searching number is P15531, and gene annotation is Nucleoside diphosphate kinase A；

134) searching number is P14550, and gene annotation is Alcohol dehydrogenase [NADP (+)]；

135) searching number is Q13421, gene annotation Mesothelin；

136) searching number is Q01853, and gene annotation is Transitional endoplasmic reticulum ATPase；

137) searching number is P10909, gene annotation Clusterin；

138) searching number is P36871, gene annotation Phosphoglucomutase-1；

139) searching number is P53396, and gene annotation is ATP-citrate synthase；

140) searching number is P14314, and gene annotation is Glucosidase 2subunit beta；

141) searching number is P39687, and gene annotation is Acidic leucine-rich nuclear phosphoprotein 32family member A；

142) searching number is Q8NC60, and gene annotation is Nitric oxide-associated protein 1；

143) searching number is P49411, and gene annotation is Elongation factor Tu, mitochondrial；

144) searching number is P29373, and gene annotation is Cellular retinoic acid-binding protein 2；

145) searching number is P52565, and gene annotation is Rho GDP-dissociation inhibitor 1；

146) searching number is P45877, and gene annotation is Peptidyl-prolyl cis-trans isomerase C；

147) searching number is O43707, gene annotation Alpha-actinin-4；

148) searching number is P18827, gene annotation Syndecan-1；

149) searching number is Q15782, and gene annotation is Chitinase-3-like protein 2；

150) searching number is P13693, and gene annotation is Translationally-controlled tumor protein；

151) searching number is P17066, and gene annotation is Heat shock 70kDa protein 6；

152) searching number is P28066, and gene annotation is Proteasome subunit alpha type-5；

153) searching number is P00558, and gene annotation is Phosphoglycerate kinase 1.

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 protein in liver cancer syndrome of deficiency of kidney yin White marker object is that the early stage diagnosis and treatment of liver cancer syndrome of deficiency of kidney yin, postoperative recurrence, transfer and Observation On The Prognosis foundation are a kind of noninvasive, easy, fast Prompt practical 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 deficiency of kidney yin biological marker analyte detection based on sialoprotein matter group, specific to walk It is rapid as follows：

(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.

(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 marks are 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 deficiency of kidney yin and normal health Crowd compares, and quantitatively detects the differential protein expression situation in saliva, while is further screening and liver cancer syndrome of deficiency of kidney yin syndrome Relevant protein or protein group provide research reference, from the syndrome essence of protein level research liver cancer syndrome of deficiency of kidney yin.

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

Syndrome of deficiency of kidney yin diagnostic criteria：

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 the study subject of participation can be coordinated.More than 3 must all meet and can just include.

1. 1.1.3 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 uniformity 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 deficiency of kidney yin trouble for meeting diagnosis and inclusion criteria is collected in this research from 04 month -2015 years in December, 2014 altogether Person 11, all cases derive from Hunan Provincial Tumour Hospital's gastroduodenal pancreatic surgery, are made a definite diagnosis through pathologic finding.Patient 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, well-differentiated adenocarcinoma 1, middle differentiation gland cancer 2, other types 14).Normal group 45, wherein man 28, female 17,23-72 years old ages, the median age 52 years old, both from the attached First Hospital physical examination section health of Hunan University of Traditional Chinese Medicine Volunteer.The research approach meets human trial ethical standard, and obtains the approval of Ethics Committee, and subject is tested It is preceding to have known, and obtain written consent.

1.3 packet design

Liver cancer syndrome of deficiency of kidney yin group VS Normal groups, to reduce experimental error, liver cancer syndrome of deficiency of kidney yin group and Normal group It is each to carry out 2 groups of technologies repetitions.

1.4 key instruments and reagent consumptive material

1.4.1 key instrument (table 1)

1 key instrument of table

Instrument title	Manufacturer	Model
			Turbula shaker	Its woods Bell's instrument manufacturing Co., Ltd of Haimen City	QL-901
Centrifuge	Thermo	PICO17
			Ultrasonic cell disruption instrument	Nanjing Xian Ou instrument manufacturings Co., Ltd	XO
Microplate reader	Termo	MultikanMK3
			Constant temperature incubates bath	Pudong, Shanghai Rong Feng scientific instrument Co., Ltd	HH.S4
Vacuum freeze drier	Thermo	SPD2010-230
			Highly effective liquid phase chromatographic system	BeiJing PuYuanJing power Science Co., Ltd	RIGOLL-3000
Mass spectrometer system	ABI	5600

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 problem.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 scope, 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 relation 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 mark

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 mark 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 deficiency of kidney yin group -1	119
4	Liver cancer syndrome of deficiency of kidney yin group -2	121

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. separated (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 separates 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 the component that high pH reverse phase separations obtain 20 μ l2% methanol, the redissolution of 0.1% formic acid.

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. separating 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 Scope：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 deficiency of kidney yin and Normal group.It is opposite and exhausted using isotope marks Quantitative (iTRAQ) technology identifies the full histone of liver cancer kidney-yin deficiency patient's saliva and quantitative analysis, tentatively obtain liver The protein groups difference expression atlas of cancer syndrome of deficiency of kidney yin patient.Quantitative proteomics result of study is shown：Using iTRAQ technologies 1326 albumen are identified altogether；Compared with Normal group, (1.5 times of the significant difference albumen identified in liver cancer syndrome of deficiency of kidney yin More than differential expression) totally 357, wherein upregulated protein 204, down-regulation protein 153.

Differential protein is listed as follows：Upregulated protein is as follows：1) searching number is Q96JC1, gene annotation Vam6/Vps39- like protein；

2) searching number is O00755, and gene annotation is Protein Wnt-7a；

3) searching number is P06702, and gene annotation is Protein S100-A9；

5) searching number is P05109, and gene annotation is Protein S100-A8；

6) searching number is Q8NDA2, gene annotation Hemicentin-2；

7) searching number is Q7Z589, and gene annotation is Protein EMSY；

14) searching number is P05164, gene annotation Myeloperoxidase；

17) searching number is O75629, and gene annotation is Protein CREG1；

19) searching number is P02795, gene annotation Metallothionein-2；

22) searching number is P02792, and gene annotation is Ferritin light chain；

24) searching number is P02771, gene annotation Alpha-fetoprotein；

25) searching number is Q16623, gene annotation Syntaxin-1A；

30) searching number is Q9HD89, gene annotation Resistin；

31) searching number is P03973, gene annotation Antileukoproteinase；

33) searching number is P01040, gene annotation Cystatin-A；

41) searching number is P08246, and gene annotation is Neutrophil elastase；

42) searching number is P20160, gene annotation Azurocidin；

47) searching number is P29034, and gene annotation is Protein S100-A2；

50) searching number is Q9Y6V0, and gene annotation is Protein piccolo；

57) searching number is Q14210, and gene annotation is Lymphocyte antigen 6D；

58) searching number is P12883, gene annotation Myosin-7；

63) searching number is Q9UBW5, and gene annotation is Bridging integrator 2；

64) searching number is P02788, gene annotation Lactotransferrin；

66) searching number is P08236, gene annotation Beta-glucuronidase；

67) searching number is O60635, gene annotation Tetraspanin-1；

73) searching number is P06748, gene annotation Nucleophosmin；

74) searching number is O75578, and gene annotation is Integrin alpha-10；

75) searching number is P59665, and gene annotation is Neutrophil defensin 1；

76) searching number is P02741, and gene annotation is C-reactive protein；

77) searching number is Q8N6Q3, gene annotation CD177antigen；

78) searching number is P46939, gene annotation Utrophin；

81) searching number is P00738, gene annotation Haptoglobin；

86) searching number is P07602, gene annotation Prosaposin；

89) searching number is P02760, and gene annotation is Protein AMBP；

96) searching number is P28799, gene annotation Granulins；

105) searching number is O95445, and gene annotation is Apolipoprotein M；

113) searching number is P02794, and gene annotation is Ferritin heavy chain；

118) searching number is Q9H244, and gene annotation is P2Y purinoceptor 12；

127) searching number is Q7RTS6, gene annotation Otopetrin-2；

131) searching number is P50995, and gene annotation is Annexin A11；

135) searching number is P06870, gene annotation Kallikrein-1；

137) searching number is Q9BPV8, and gene annotation is P2Y purinoceptor 13；

138) searching number is P13987, gene annotation CD59glycoprotein；

141) searching number is P09525, and gene annotation is Annexin A4；

142) searching number is O15400, gene annotation Syntaxin-7；

143) searching number is P31949, and gene annotation is Protein S100-A11；

146) searching number is O43866, gene annotation CD5antigen-like；

151) searching number is P98088, gene annotation Mucin-5AC；

153) searching number is P35241, gene annotation Radixin；

155) searching number is P25774, and gene annotation is Cathepsin S；

156) searching number is P08603, and gene annotation is Complement factor H；

160) searching number is P19957, gene annotation Elafin；

164) searching number is P05107, and gene annotation is Integrin beta-2；

166) searching number is O43768, gene annotation Alpha-endosulfine；

168) searching number is P02790, gene annotation Hemopexin；

170) searching number is P31151, and gene annotation is Protein S100-A7；

175) searching number is O75635, and gene annotation is Serpin B7；

177) searching number is P08758, and gene annotation is Annexin A5；

179) searching number is O15031, gene annotation Plexin-B2；

186) searching number is Q86SG5, and gene annotation is Protein S100-A7A；

188) searching number is P01023, gene annotation Alpha-2-macroglobulin；

198) searching number is P00734, gene annotation Prothrombin；

199) searching number is Q99584, and gene annotation is Protein S100-A13；

Down-regulation protein is as follows：

2) searching number is O60814, and gene annotation is Histone H2B type 1-K；

3) searching number is Q06828, gene annotation Fibromodulin；

4) searching number is P02808, gene annotation Statherin；

6) searching number is Q8IVL1, and gene annotation is Neuron navigator 2；

8) searching number is P80303, gene annotation Nucleobindin-2；

9) searching number is Q96A08, and gene annotation is Histone H2B type 1-A；

11) searching number is P04745, and gene annotation is Alpha-amylase 1；

12) searching number is Q8IUE6, and gene annotation is Histone H2A type 2-B；

13) searching number is Q9H2E6, gene annotation Semaphorin-6A；

16) searching number is P35754, gene annotation Glutaredoxin-1；

24) searching number is P32243, and gene annotation is Homeobox protein OTX2；

27) searching number is Q92520, and gene annotation is Protein FAM3C；

28) searching number is P23280, and gene annotation is Carbonic anhydrase 6；

32) searching number is Q14118, gene annotation Dystroglycan；

33) searching number is P0C0S5, and gene annotation is Histone H2A.Z；

34) searching number is P16401, and gene annotation is Histone H1.5；

35) searching number is P02652, and gene annotation is Apolipoprotein A-II；

38) searching number is P16870, and gene annotation is Carboxypeptidase E；

45) searching number is O43852, gene annotation Calumenin；

52) searching number is O75695, and gene annotation is Protein XRP2；

54) searching number is P01036, gene annotation Cystatin-S；

57) searching number is Q04917, and gene annotation is 14-3-3protein eta；

58) searching number is P58499, and gene annotation is Protein FAM3B；

59) searching number is P30085, and gene annotation is UMP-CMP kinase；

62) searching number is P02647, and gene annotation is Apolipoprotein A-I；

65) searching number is P62805, and gene annotation is Histone H4；

67) searching number is Q05315, gene annotation Galectin-10；

69) searching number is P15515, gene annotation Histatin-1；

71) searching number is P28325, gene annotation Cystatin-D；

72) searching number is P63313, and gene annotation is Thymosin beta-10；

79) searching number is P15289, and gene annotation is Arylsulfatase A；

80) searching number is O00602, gene annotation Ficolin-1；

85) searching number is Q96FV2, gene annotation Secernin-2；

86) searching number is Q96BQ1, and gene annotation is Protein FAM3D；

87) searching number is Q9NRS6, and gene annotation is Sorting nexin-15；

88) searching number is P05089, gene annotation Arginase-1；

92) searching number is P60709, gene annotation Actin, cytoplasmic 1；

93) searching number is P30041, gene annotation Peroxiredoxin-6；

94) searching number is P20962, gene annotation Parathymosin；

96) searching number is P68431, and gene annotation is Histone H3.1；

98) searching number is P07737, gene annotation Profilin-1；

100) searching number is P46108, and gene annotation is Adapter molecule crk；

101) searching number is O00584, and gene annotation is Ribonuclease T2；

104) searching number is Q02413, gene annotation Desmoglein-1；

111) searching number is Q8TAX7, gene annotation Mucin-7；

112) searching number is P0C0L4, and gene annotation is Complement C4-A；

113) searching number is Q96C23, and gene annotation is Aldose 1-epimerase；

116) searching number is Q15493, gene annotation Regucalcin；

118) searching number is Q9UHA7, gene annotation Interleukin-36alpha；

119) searching number is Q9UBX7, gene annotation Kallikrein-11；

120) searching number is P23528, gene annotation Cofilin-1；

122) searching number is Q8NI35, and gene annotation is InaD-like protein；

125) searching number is P01037, gene annotation Cystatin-SN；

127) searching number is Q8IWF6, and gene annotation is Protein DENND6A；

135) searching number is Q13421, gene annotation Mesothelin；

137) searching number is P10909, gene annotation Clusterin；

138) searching number is P36871, gene annotation Phosphoglucomutase-1；

139) searching number is P53396, and gene annotation is ATP-citrate synthase；

147) searching number is O43707, gene annotation Alpha-actinin-4；

148) searching number is P18827, gene annotation Syndecan-1；

Plurality of differential protein (such as Prothrombin (F2), Alpha-2-macroglobulin (A2M), Alpha- actinin-4(ACTN4)、Prostaglandin G/H synthase 2(PTGS2)、BH3-interacting domain Death agonist (BID) etc.) some important metabolic pathways are taken part in, it may be close with the generation, development, prognosis of liver cancer It is related.

ITRAQ labelling techniques can separate 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 deficiency of kidney yin that may be valuable are early Phase saliva hurtless measure molecular diagnostics biological marker；Further disclose the substance base of liver cancer syndrome of deficiency of kidney yin molecular group level Plinth and scientific meaning are laid a good foundation for its further occurrence and development mechanism and its biomarker research.

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 on the premise of this patent objective not be departed from the knowledge that one skilled in the relevant art possesses Various changes can be made.

Claims

1. the method for the liver cancer syndrome of deficiency of kidney yin biological marker analyte detection based on sialoprotein matter group, which is characterized in that specific step It is rapid 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 relation 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 separated 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 the component that high pH reverse phase separations obtain with 20 μ l2% methanol, 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 separated, 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 nephrasthenia syndrome 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 nephrasthenia syndrome 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 Q96JC1, and gene annotation is Vam6/Vps39-like protein；

2) searching number is O00755, and gene annotation is Protein Wnt-7a；

3) searching number is P06702, and gene annotation is Protein S100-A9；

5) searching number is P05109, and gene annotation is Protein S100-A8；

6) searching number is Q8NDA2, gene annotation Hemicentin-2；

7) searching number is Q7Z589, and gene annotation is Protein EMSY；

12) searching number is P0CG06, and gene annotation is Ig lambda-3 chain C regions；

14) searching number is P05164, gene annotation Myeloperoxidase；

17) searching number is O75629, and gene annotation is Protein CREG1；

19) searching number is P02795, gene annotation Metallothionein-2；

22) searching number is P02792, and gene annotation is Ferritin light chain；

24) searching number is P02771, gene annotation Alpha-fetoprotein；

25) searching number is Q16623, gene annotation Syntaxin-1A；

30) searching number is Q9HD89, gene annotation Resistin；

31) searching number is P03973, gene annotation Antileukoproteinase；

33) searching number is P01040, gene annotation Cystatin-A；

41) searching number is P08246, and gene annotation is Neutrophil elastase；

42) searching number is P20160, gene annotation Azurocidin；

46) searching number is Q9H7L9, and gene annotation is Sin3 histone deacetylase corepressor complex component SDS3；

47) searching number is P29034, and gene annotation is Protein S100-A2；

49) searching number is Q8NCM8, gene annotation is 2 heavy chain 1 of Cytoplasmic dynein；

50) searching number is Q9Y6V0, and gene annotation is Protein piccolo；

57) searching number is Q14210, and gene annotation is Lymphocyte antigen 6D；

58) searching number is P12883, gene annotation Myosin-7；

63) searching number is Q9UBW5, and gene annotation is Bridging integrator 2；

64) searching number is P02788, gene annotation Lactotransferrin；

66) searching number is P08236, gene annotation Beta-glucuronidase；

67) searching number is O60635, gene annotation Tetraspanin-1；

73) searching number is P06748, gene annotation Nucleophosmin；

74) searching number is O75578, and gene annotation is Integrin alpha-10；

75) searching number is P59665, and gene annotation is Neutrophil defensin 1；

76) searching number is P02741, and gene annotation is C-reactive protein；

77) searching number is Q8N6Q3, gene annotation CD177antigen；

78) searching number is P46939, gene annotation Utrophin；

81) searching number is P00738, gene annotation Haptoglobin；

86) searching number is P07602, gene annotation Prosaposin；

89) searching number is P02760, and gene annotation is Protein AMBP；

92) searching number is P49221, and gene annotation is Protein-glutamine gamma-glutamyltransferase 4；

96) searching number is P28799, gene annotation Granulins；

105) searching number is O95445, and gene annotation is Apolipoprotein M；

106) searching number is P04839, and gene annotation is Cytochrome b-245 heavy chain；

113) searching number is P02794, and gene annotation is Ferritin heavy chain；

114) searching number is P61457, and gene annotation is Pterin-4-alpha-carbinolamine dehydratase；

118) searching number is Q9H244, and gene annotation is P2Y purinoceptor 12；

127) searching number is Q7RTS6, gene annotation Otopetrin-2；

131) searching number is P50995, and gene annotation is Annexin A11；

135) searching number is P06870, gene annotation Kallikrein-1；

136) searching number is P01877, and gene annotation is Ig alpha-2 chain C region；

137) searching number is Q9BPV8, and gene annotation is P2Y purinoceptor 13；

138) searching number is P13987, and gene annotation is CD59 glycoprotein；

141) searching number is P09525, and gene annotation is Annexin A4；

142) searching number is O15400, gene annotation Syntaxin-7；

143) searching number is P31949, and gene annotation is Protein S100-A11；

146) searching number is O43866, gene annotation CD5antigen-like；

151) searching number is P98088, gene annotation Mucin-5AC；

153) searching number is P35241, gene annotation Radixin；

155) searching number is P25774, and gene annotation is Cathepsin S；

156) searching number is P08603, and gene annotation is Complement factor H；

160) searching number is P19957, gene annotation Elafin；

164) searching number is P05107, and gene annotation is Integrin beta-2；

166) searching number is O43768, gene annotation Alpha-endosulfine；

167) searching number is P16219, and gene annotation is Short-chain specific acyl-CoA dehydrogenase,mitochondrial；

168) searching number is P02790, gene annotation Hemopexin；

170) searching number is P31151, and gene annotation is Protein S100-A7；

175) searching number is O75635, and gene annotation is Serpin B7；

177) searching number is P08758, and gene annotation is Annexin A5；

179) searching number is O15031, gene annotation Plexin-B2；

181) searching number is P01861, and gene annotation is Ig gamma-4 chain C region；

186) searching number is Q86SG5, and gene annotation is Protein S100-A7A；

187) searching number is Q9Y608, and gene annotation is Leucine-rich repeat flightless-interacting protein 2

188) searching number is P01023, gene annotation Alpha-2-macroglobulin；

194) searching number is Q6PCB0, and gene annotation is von Willebrand factor A domain-containing protein 1；

198) searching number is P00734, gene annotation Prothrombin；

199) searching number is Q99584, and gene annotation is Protein S100-A13；

200) searching number is P10644, and gene annotation is cAMP-dependent protein kinase type I-alpha regulatory subunit；

203) searching number is P01859, and gene annotation is Ig gamma-2 chain C region；

Down-regulation protein is as follows：

2) searching number is O60814, and gene annotation is Histone H2B type 1-K；

3) searching number is Q06828, gene annotation Fibromodulin；

4) searching number is P02808, gene annotation Statherin；

6) searching number is Q8IVL1, and gene annotation is Neuron navigator 2；

8) searching number is P80303, gene annotation Nucleobindin-2；

9) searching number is Q96A08, and gene annotation is Histone H2B type 1-A；

10) searching number is Q9BYB0, and gene annotation is SH3 and multiple ankyrin repeat domains protein 3；

11) searching number is P04745, and gene annotation is Alpha-amylase 1；

12) searching number is Q8IUE6, and gene annotation is Histone H2A type 2-B；

13) searching number is Q9H2E6, gene annotation Semaphorin-6A；

16) searching number is P35754, gene annotation Glutaredoxin-1；

24) searching number is P32243, and gene annotation is Homeobox protein OTX2；

26) searching number is Q9H299, and gene annotation is SH3 domain-binding glutamic acid-rich-like protein 3；

27) searching number is Q92520, and gene annotation is Protein FAM3C；

28) searching number is P23280, and gene annotation is Carbonic anhydrase 6；

30) searching number is P22626, and gene annotation is Heterogeneous nuclear ribonucleoproteins A2/ B1；

32) searching number is Q14118, gene annotation Dystroglycan；

33) searching number is P0C0S5, and gene annotation is Histone H2A.Z；

34) searching number is P16401, and gene annotation is Histone H1.5；

35) searching number is P02652, and gene annotation is Apolipoprotein A-II；

38) searching number is P16870, and gene annotation is Carboxypeptidase E；

45) searching number is O43852, gene annotation Calumenin；

52) searching number is O75695, and gene annotation is Protein XRP2；

54) searching number is P01036, gene annotation Cystatin-S；

57) searching number is Q04917, and gene annotation is 14-3-3 protein eta；

58) searching number is P58499, and gene annotation is Protein FAM3B；

59) searching number is P30085, and gene annotation is UMP-CMP kinase；

60) searching number is Q9P0M2, and gene annotation is 7 isoform gamma of A-kinase anchor protein；

62) searching number is P02647, and gene annotation is Apolipoprotein A-I；

64) searching number is Q96DA0, and gene annotation is 16 homolog B of Zymogen granule protein；

65) searching number is P62805, and gene annotation is Histone H4；

67) searching number is Q05315, gene annotation Galectin-10；

69) searching number is P15515, gene annotation Histatin-1；

71) searching number is P28325, gene annotation Cystatin-D；

72) searching number is P63313, and gene annotation is Thymosin beta-10；

79) searching number is P15289, and gene annotation is Arylsulfatase A；

80) searching number is O00602, gene annotation Ficolin-1；

82) searching number is P04406, and gene annotation is Glyceraldehyde-3-phosphate dehydrogenase；

85) searching number is Q96FV2, gene annotation Secernin-2；

86) searching number is Q96BQ1, and gene annotation is Protein FAM3D；

87) searching number is Q9NRS6, and gene annotation is Sorting nexin-15；

88) searching number is P05089, gene annotation Arginase-1；

92) searching number is P60709, gene annotation Actin, cytoplasmic 1；

93) searching number is P30041, gene annotation Peroxiredoxin-6；

94) searching number is P20962, gene annotation Parathymosin；

96) searching number is P68431, and gene annotation is Histone H3.1；

98) searching number is P07737, gene annotation Profilin-1；

100) searching number is P46108, and gene annotation is Adapter molecule crk；

101) searching number is O00584, and gene annotation is Ribonuclease T2；

104) searching number is Q02413, gene annotation Desmoglein-1；

111) searching number is Q8TAX7, gene annotation Mucin-7；

112) searching number is P0C0L4, and gene annotation is Complement C4-A；

113) searching number is Q96C23, and gene annotation is Aldose 1-epimerase；

116) searching number is Q15493, gene annotation Regucalcin；

118) searching number is Q9UHA7, gene annotation Interleukin-36alpha；

119) searching number is Q9UBX7, gene annotation Kallikrein-11；

120) searching number is P23528, gene annotation Cofilin-1；

122) searching number is Q8NI35, and gene annotation is InaD-like protein；

125) searching number is P01037, gene annotation Cystatin-SN；

127) searching number is Q8IWF6, and gene annotation is Protein DENND6A；

135) searching number is Q13421, gene annotation Mesothelin；

137) searching number is P10909, gene annotation Clusterin；

138) searching number is P36871, gene annotation Phosphoglucomutase-1；

139) searching number is P53396, and gene annotation is ATP-citrate synthase；

141) searching number is P39687, and gene annotation is Acidic leucine-rich nuclear phosphoprotein 32 family member A；

147) searching number is O43707, gene annotation Alpha-actinin-4；

148) searching number is P18827, gene annotation Syndecan-1；