CN113740544A - Method for identifying biomarkers of prolactin protein existing form of human pituitary tissue - Google Patents

Abstract

The invention provides a method for identifying a biomarker of the existence form of prolactin protein in human pituitary tissue, which comprises the following steps: collecting pituitary adenoma and normal pituitary tissue samples, respectively extracting tissue proteins, scanning a visual PVDF film and a bidirectional gel into a digital image through bidirectional gel electrophoresis and western blot silver staining, digesting and purifying corresponding bidirectional gel protein points by trypsin, and identifying the difference of the existence form proportion of the prolactin protein of the pituitary tissue among different pituitary tumor subtypes through mass spectrum identification and bioinformatics analysis. The invention identifies the existence forms of prolactin proteins in human pituitary tissues and the differential expression profiles of the prolactin proteins in different pituitary tumors, and is favorable for developing the medicaments for blocking the prolactin signal path for clinical treatment.

Description

Method for identifying biomarkers of prolactin protein existing form of human pituitary tissue

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a method for identifying a biomarker of the existence form of prolactin protein in human pituitary tissue.

Background

Pituitary tumors are one of the most common primary intracranial tumors, accounting for 17% of intracranial tumors. Prolactin (PRL) which is abnormally secreted by pituitary tumors can cause clinical diseases such as hyperprolactinemia. Despite various therapeutic approaches such as drug therapy and surgical resection, a series of PRL-related diseases caused by pituitary tumors remain important clinical problems. Therefore, the search for Prolactin protein-presented form (PRLP) biomarkers of pituitary tumors and PRLP biomarkers of related Prolactin-related diseases is one of the key means for the management of pituitary tumors and Prolactin-related diseases.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a method for identifying the existence forms of prolactin proteins in human pituitary tissue aiming at the defects of the prior art, and the method is used for identifying the existence forms of prolactin proteins in human pituitary tissue and the differential expression profiles of the prolactin proteins in different pituitary tumor subtypes, and is favorable for developing a medicament for blocking a PRL signal pathway for clinical treatment.

In order to solve the technical problems, the invention adopts the technical scheme that: a method for identifying a biomarker for the presence of prolactin protein in human pituitary tissue, the method comprising:

s1, collecting pituitary adenoma and normal pituitary tissue samples, respectively cracking the tissues and extracting to respectively obtain pituitary adenoma tissue protein and normal pituitary tissue protein;

s2, dividing each group of the pituitary adenoma tissue protein obtained in the S1 and the normal pituitary tissue protein into two parts, respectively carrying out two-dimensional gel electrophoresis, carrying out the two-dimensional gel electrophoresis on the pituitary adenoma tissue protein to obtain a protein-containing two-dimensional gel a and a protein-containing two-dimensional gel A, and carrying out the two-dimensional gel electrophoresis on the normal pituitary tissue protein to obtain a protein-containing two-dimensional gel B and a protein-containing two-dimensional gel B;

s3, respectively carrying out western blotting on the protein-containing bidirectional gel a and the protein-containing bidirectional gel b obtained in S2, transferring the proteins on the bidirectional gels to polyvinylidene fluoride membranes to respectively obtain a PVDF membrane a and a PVDF membrane b, then the PVDF membrane a and the PVDF membrane b are respectively immersed in the sealing solution, shaken and sealed on a decoloring shaking table for 1 hour and then taken out, then soaking the mixture into primary antibody solution, shaking and incubating for 1h, washing and decolorizing with PBST solution for 4 times on a decolorizing shaker for 15min each time, washing with double distilled water for 2 times, then immersing the membrane into a second antibody solution, shaking and incubating for 1h, washing and decoloring for 4 times by using a PBST solution on a decoloring shaking table, washing and decoloring for 15min each time, and developing the treated PVDF membrane a and PVDF membrane b by using 5-bromo-4-chloro-3-indolyl phosphoric acid to obtain a visualized PVDF membrane a and a visualized PVDF membrane b;

the primary antibody solution is prepared by mixing a primary antibody solution and a secondary antibody solution in a volume ratio of 1: 1000 rabbit anti-human prolactin antibody and 0.3% bovine serum albumin-PBST solution;

the volume ratio of the secondary antibody solution is 1: 4000 parts of mixed solution of goat anti-rabbit alkaline phosphate IgG and bovine serum albumin-PBST solution with the mass fraction of 0.3%; the PBST solution is a phosphate buffer solution containing 0.2% of Tween 20 by mass;

s4, displaying the protein-containing bidirectional gel A and the protein-containing bidirectional gel B obtained in the S2 by a silver staining method to obtain a stained bidirectional gel A and a stained bidirectional gel B; displaying the protein-containing bidirectional gel a and the protein-containing bidirectional gel b subjected to western blotting in S3 by using a silver staining method to obtain a stained bidirectional gel a and a stained bidirectional gel b;

s5, scanning the visual PVDF film a and B obtained in S3, the dyed bidirectional gel A, the dyed bidirectional gel B, the dyed bidirectional gel a and the dyed bidirectional gel B obtained in S4 into digitized images, and introducing the digitized images into PDQuest^TMQuantifying the volume of protein spots in the software for analyzing the bidirectional gel image, and matching the protein spots which are positive in immunity with the protein spots of the corresponding dyed bidirectional gel a and the dyed bidirectional gel b;

s6, performing enzymolysis on the bidirectional gel protein spots corresponding to the western blot spots which are positive in the visual PVDF membrane a in S3 in the gel spots of the dyed bidirectional gel A and the dyed bidirectional gel a obtained in S4 by using trypsinThe proteins in the spot were extracted and the peptide mixture was digested with trypsin and then treated with ZipTipC₁₈Purifying the microcolumn to obtain a purified trypsin enzymolysis peptide mixture a;

subjecting the gel spots of the stained bidirectional gel B and the stained bidirectional gel B obtained in S4, which correspond to the immunopositive western blot spots of the visualized PVDF membrane B obtained in S3, to trypsin enzymolysis, extracting a trypsin enzymolysis peptide mixture, and subjecting the trypsin enzymolysis peptide mixture to ZipTipC₁₈Purifying the microcolumn to obtain a purified trypsin enzymolysis peptide mixture b;

s7, carrying out MALDI-TOF-MS analysis on the purified trypsin enzymolysis peptide mixture a and the purified trypsin enzymolysis peptide mixture b obtained in S6 to obtain peptide fingerprint PMF data, carrying out LC-ESI-MS/MS analysis to obtain MS/MS data or carrying out MALDI-TOF-TOF-MS/MS analysis to obtain PMF data and MS/MS data, and obtaining original mass spectrum data;

s8, inquiring the original mass spectrum data obtained in S7 in a SwissProt human protein database, identifying human prolactin protein, comparing the original mass spectrum data obtained in each glue dot with a theoretical mass spectrum peak of the human prolactin protein, and identifying whether signal peptide is absent; predicting a prolactin phosphorylation site, an N-glycosylation site and an O-glycosylation site of human pituitary tissue by a NetPhos3.1 server, a NetNGlyc 1.0server and a NetOGlyc 4.0server respectively;

s9, analyzing the difference of the existence form ratio of the prolactin protein of the pituitary tissues among different pituitary tumor subtypes by using SPSS 22 software chi-square test.

Preferably, the blocking solution in S3 is a mixture of 0.3% bovine serum albumin-PBST solution and 0.1% sodium azide-PBST solution in a volume ratio of 1: 1.

Compared with the prior art, the invention has the following advantages:

the present invention is based on immunoblotting (Western blot) with two-dimensional gel electrophoresis (2DGE) in combination with anti-hPRL antibodies, Mass Spectrometry (MS) and bioinformatics to identify the presence of human pituitary hPRL proteins and their useDifferential expression profiles in different pituitary tumors. 6 forms of hPRL protein were identified in the human pituitary (V1, V2, V3, V4, V5, V6), including 2 forms of hPRL protein not detected by Western blotting (V2 and V3). 6 hPRL proteins exist in 5 pituitary tumor subtypes (NF-, FSH)⁺-,LH⁺-,FSH⁺/LH⁺-,and PRL⁺Pituitary tumors) were significantly different. Furthermore, the 6 forms of hPRL protein present in human pituitary are derived from hPRL hormone precursors with different post-translational modifications of 227 amino acids (1-227), such as deamidation, glycosylation and phosphorylation, rather than mature hPRL with 199 amino acids (29-227), and these findings provide new insights into the function and mechanism of human pituitary hPRL and its related diseases, as well as its potential clinical value in pituitary tumors. The signal pathway involved in the existence form of hPRL protein may elucidate the biological functions of different existence forms of hPRL protein and its potential clinical value, and may be used in developing medicine for blocking PRL signal pathway.

The present invention will be described in further detail with reference to the accompanying drawings and examples.

Drawings

FIG. 1 is a silver stain of the prolactin protein form of human pituitary tissue of the present invention (n-6, pI: isoelectric point).

FIG. 2 is a Western blotting graph (A) showing the presence of prolactin protein in human pituitary tissue of the present invention and a silver staining graph (B) after Western blotting (pI: isoelectric point).

FIG. 3A peptide fragment derived from prolactin protein form V6 according to the invention¹¹⁸SWNEPLYHLVTEVR¹³¹MS/MS spectra of (1).

Detailed Description

Example 1

The method for identifying the biomarker of the prolactin protein existing form of human pituitary tissue comprises the following steps:

s1, collecting pituitary adenoma and normal pituitary tissue samples, respectively cracking the tissues and extracting to respectively obtain pituitary adenoma tissue protein and normal pituitary tissue protein;

human control group normal pituitary tissue samples were postmortem tissue, obtained from the national centers for disease communication and the medical center in menfeis, and approved by the university of tennessee for health. Human pituitary adenoma tissue was obtained from the Emeric university Hospital, approved by the university, and all tissues were removed and stored in liquid nitrogen until processed;

the specific protein extraction method comprises the following steps:

each tissue sample (about 600mg) was washed with 0.9% by mass sodium chloride solution (3ml, 5 times) to completely remove blood, and the tissue was finely ground in liquid nitrogen, then a reagent was added to mix well to give a mixture, and the mixture was vortexed on ice (2h) and then centrifuged (15,000 xg, 15min,4 ℃). Collecting supernatant, and determining protein concentration (Bio-Rad) with Bio-Rad 2D quantitative kit, wherein the supernatant is obtained pituitary adenoma tissue protein and normal pituitary tissue protein; the reagent consists of the following materials in amount: 5mL of protein extraction buffer containing 2mL of thiourea, 7mol/L of urea, 40g/L of 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt (CHAPS), 100mmol/L of Dithiothreitol (DTT), and 3-10NL 5mol/L of solid phase pH gradient (IPG) buffer, and a trace amount of bromophenol blue was added.

S2, dividing each group of the pituitary adenoma tissue protein obtained in the S1 and the normal pituitary tissue protein into two parts, respectively carrying out two-dimensional gel electrophoresis, carrying out the two-dimensional gel electrophoresis on the pituitary adenoma tissue protein to obtain a protein-containing two-dimensional gel a and a protein-containing two-dimensional gel A, and carrying out the two-dimensional gel electrophoresis on the normal pituitary tissue protein to obtain a protein-containing two-dimensional gel B and a protein-containing two-dimensional gel B;

the specific method of the two-dimensional gel electrophoresis comprises the following steps:

(1) first isoelectric focusing:

isoelectric focusing was performed using a pre-fabricated IPG strip (pH 3-10NL, 180X 3X 0.5mm) and 18cm IPG strip slot on an IPGphor instrument (GE Healthcare). Prior to isoelectric focusing, a defined amount (350. mu.L) of protein sample solution was loaded onto the IPG gel chamber, the IPG gel strip was placed and hydrated for 18 hours. Isoelectric focusing was performed at 25 ℃ under the following conditions: one gradient reaches 25V and runs for 1 hour and the voltage time product is 125Vh, one gradient reaches 1000V and runs for 1 hour and the voltage time product is 500Vh, one gradient reaches 8000V and runs for 1 hour and the voltage time product is 4000Vh, one step reaches 8000V and runs for 4 hours and the voltage time product is 32000Vh, one step reaches 500V and runs for 0.5 hour and reaches 250Vh, the voltage time product 36875Vh is reached totally, and the operation is 7.5 hours. The IPG strip was removed and the mineral oil on the back of the plastic was blotted off.

(2)SDS-PAGE：

After isoelectric focusing, an Ettan DALT II system (GE Healthcare, 12 blocks of gel can be run at a time) was used. A12% PAGE gel (250X 215X 1.0mm) was added to Ettan DALTsix (high resolution medium throughput vertical electrophoresis system). This separation gel composition is as follows: 75mL of 1.5mol/L Tris-HCl pH 8.8, 90mL of 400g/L acrylamide-bisacrylamide solution (29: 1 w/w: 3.3% w/w crosslinking ratio), 3mL of 10% by weight ammonium persulfate solution, and 150mL of deionized distilled water ((ddH)₂O),50 μ L of tetramethylethylenediamine, the IPG gel strip was equilibrated in 15mL of reducing equilibration buffer consisting of the following raw materials for 15 min: 375mmol/L Tris-HCl pH 8.8, SDS solution with the mass concentration of 20g/L, glycerol solution with the volume fraction of 20 percent and a trace amount of bromophenol blue. The IPG strip was then equilibrated in 15mL of an alkylation equilibration solution containing 2.5% w/v of iodoacetamide. The equilibrated IPG gel strip was sealed on top of the separation gel with a boiling 1% w/v agarose containing solution. The two-dimensional electrophoresis was carried out at 25 ℃ under a constant pressure (250V,360min) in 25L Tris-glycine-SDS buffer containing 192mmol/L glycine, 25mmol/L Tris base (Tris hydroxymethyl aminomethane) and 1% w/V SDS.

S3, the protein-containing bidirectional gel a and the protein-containing bidirectional gel b obtained in S2 were removed, western blotting was performed, and the proteins on the bidirectional gels were transferred to a polyvinylidene fluoride (PVDF) membrane (0.8 mA/cm) using a semidry transfer apparatus (Amersham Pharmacia Biotech Nova Blot)²Lasting for 1 hour and 20 minutes, PVDF films a and PV are obtained respectivelyImmersing the PVDF membrane a and the PVDF membrane b into a sealing solution respectively, shaking on a decoloring shaking table, taking out after sealing for 1h, immersing the PVDF membrane a and the PVDF membrane b into a primary antibody solution, shaking, incubating for 1h, washing and decoloring for 4 times by using a PBST solution on the decoloring shaking table, washing and decoloring for 15min each time, washing for 2 times by using double distilled water, immersing the PVDF membrane a and the PVDF membrane b into a secondary antibody solution, shaking and incubating for 1h, washing and decoloring for 4 times by using the PBST solution on the decoloring shaking table, washing and decoloring for 15min each time, and developing the treated PVDF membrane a and the PVDF membrane b by using 5-bromo-4-chloro-3-indolyl phosphoric acid to obtain a visualized PVDF membrane a and a visualized PVDF membrane b;

the primary antibody solution is prepared by mixing a primary antibody solution and a secondary antibody solution in a volume ratio of 1: 1000 rabbit anti-human prolactin antibody and 0.3% bovine serum albumin-PBST solution;

the volume ratio of the secondary antibody solution is 1: 4000 parts of mixed solution of goat anti-rabbit alkaline phosphate IgG and bovine serum albumin-PBST solution with the mass fraction of 0.3%; the PBST solution is a phosphate buffer solution containing 0.2% of Tween 20 by mass;

the confining liquid is a mixed liquid of bovine serum albumin-PBST solution with the mass fraction of 0.3% and sodium azide-PBST solution with the mass fraction of 0.1% in a volume ratio of 1: 1;

s4, displaying the protein-containing bidirectional gel A and the protein-containing bidirectional gel B obtained in the S2 by a silver staining method to obtain a stained bidirectional gel A and a stained bidirectional gel B; displaying the protein-containing bidirectional gel a and the protein-containing bidirectional gel b subjected to western blotting in S3 by using a silver staining method to obtain a stained bidirectional gel a and a stained bidirectional gel b;

s5, scanning the visual PVDF film a and B obtained in S3, the dyed bidirectional gel A, the dyed bidirectional gel B, the dyed bidirectional gel a and the dyed bidirectional gel B obtained in S4 into digitized images, and introducing the digitized images into PDQuest^TMQuantifying the volume of protein spots in the software for analyzing the bidirectional gel image, and matching the protein spots which are positive in immunity with the protein spots of the corresponding dyed bidirectional gel a and the dyed bidirectional gel b;

s6, mixingThe gel spots of the dyed bidirectional gel A and the dyed bidirectional gel a obtained in S4 correspond to the western blot spots which are positive in immunity and are in the visualized PVDF membrane a in S3, the protein in the bidirectional gel protein spots is subjected to enzymolysis by trypsin, a trypsin enzymolysis peptide mixture is extracted, and then ZipTipC is used for enzymolysis₁₈Purifying the microcolumn to obtain a purified trypsin enzymolysis peptide mixture a;

subjecting the gel spots of the stained bidirectional gel B and the stained bidirectional gel B obtained in S4, which correspond to the immunopositive western blot spots of the visualized PVDF membrane B obtained in S3, to trypsin enzymolysis, extracting a trypsin enzymolysis peptide mixture, and subjecting the trypsin enzymolysis peptide mixture to ZipTipC₁₈Purifying the microcolumn to obtain a purified trypsin enzymolysis peptide mixture b;

s7, subjecting the purified trypsin enzymolysis peptide mixture a and the purified trypsin enzymolysis peptide mixture b obtained in S6 to MALDI-TOF-MS (matrix assisted laser desorption ionization time-of-flight mass spectrometry) analysis to obtain peptide fingerprint PMF data, LC-ESI-MS/MS (liquid chromatography electrospray tandem mass spectrometry) analysis to obtain MS/MS data or MALDI-TOF-TOF-MS/MS (matrix assisted laser desorption ionization tandem time-of-flight mass spectrometry) analysis to obtain PMF data and MS/MS data, and obtaining original mass spectrometry data to identify proteins;

MALDI-TOF-MS: analysis of those proteins digested by trypsin, MALDI-TOF-MS protonates the molecular ions [ M + H ]]⁺. Data processing software (DataExplore) was used to obtain accurate quality. Blank gel experiments were also performed to exclude mass spectral peaks derived from known contaminants (usually keratin), matrix, trypsin and other unknown contaminants. Each protein was identified by searching the Swiss-Prot database 091215(513877 sequence, 180750753 residues) using Peptidet software to obtain MALDI-TOF MS peptide fingerprinting (PMF data).

LC-ESI-MS/MS: the purified trypsin enzymolysis peptide mixture a and the trypsin enzymolysis peptide mixture b are subjected to LCQ^DecaAnd (5) analyzing by a mass spectrometer. The instrument parameters are: the protein was identified using MS/MS data from LC-ESI-Q-IT using a Swiss-Prot database with an electron multiplier of 900v, ESI (electrospray ion source) voltage of 2.0kv, capillary probe temperature of 110 ℃.

MALDI-TOF-TOF-MS/MS: and analyzing the purified trypsin enzymolysis peptide mixture a and the purified trypsin enzymolysis peptide mixture b, and analyzing the polypeptide by using MALDI-TOF-TOF-MS, wherein the mass spectrum analysis is operated in a reflection mode, the acceleration voltage is 25kv, and the m/z (mass-to-charge ratio) is 800-4000. The parent ion close to the theoretical m/z was chosen for TOF-TOF mass spectrometry, obtaining mass spectrometry data (PMF data and MS/MS data). And querying a database by using the obtained mass spectrum data to identify the protein.

S8, inquiring the original mass spectrum data obtained in S7 in a SwissProt human protein database, identifying human prolactin protein, comparing the original mass spectrum data obtained in each glue dot with a theoretical mass spectrum peak of the human prolactin protein, and identifying whether signal peptide is absent; predicting a prolactin phosphorylation site, an N-glycosylation site and an O-glycosylation site of human pituitary tissue by a NetPhos3.1 server, a NetNGlyc 1.0server and a NetOGlyc 4.0server respectively;

s9, combining the existence forms of hRPL protein identified in S7 with the volume of the two-dimensional electrophoresis gel point corresponding to the existence forms of the prolactin protein, and analyzing the difference of the proportion of the different existence forms of hPRL protein among different pituitary tumor subtypes by using SPSS 22 software chi-square test, wherein the pituitary tumor subtypes are determined by clinical pathological diagnosis.

(one) the distribution pattern of the two-dimensional electrophoresis and the expression change among different pituitary tumor subtypes of 6 human pituitary hPRL proteins:

approximately 1200 protein spots were detected in each silver stained 2D gel. Of these 6 protein spots were found to contain PRL (Swiss-Prot No. P01236) (FIG. 1). The 6 spots were identified by mass spectrometry to contain different pI-M_rPatterned hPRL, including the hPRL protein forms v1(pI 6.1; 26.0kDa), v2(pI 6.3; 26.4kDa), v3(pI 6.3; 27.9kDa), v4(pI 6.5; 26.1kDa), v5(pI 6.8; 25.9kDa) and v6(pI 6.7; 25.9 kDa). This is illustrated by the identification of the mass spectrum of v6 for hPRL as an example. By MALDI-TOF MS or MALThe tryptic peptide fragments of v6 were analyzed by DI-TOF-TOF MS. A peptide fingerprint from v6 was obtained, querying the Swiss-Prot human protein database, with 4 peptide fragments significantly matching hPRL (P01236). In addition, MS/MS analysis of the peptide fragment from v6 was also performed using MALDI-TOF-TOF MS/MS or LC-ESI-Q-IT MS/MS. Sequencing of 3 peptide fragments from v6 with MS-MS data, including⁷²YTHGRGFITK⁸¹，¹¹⁸SWNEPLYHLVTEVR¹³¹，¹⁷1ENEIYPVWSGLPSLQMADEESR¹⁹²Significantly matched the hPRL (P01236) in the Swiss-Prot human protein database. For example, peptide fragments¹¹⁸SWNEPLYHLVTEVR¹³¹The MS/MS spectrum of (A) contains a rich series of ions, including b₈,b₉,b₁₂,b₁₃,y₁,y₂,y₅,y₆,y₇,y₁₀,y₁₁And y₁₂Ions (fig. 3). Using the same procedure, 5 other hPRLs, v1-v5, were identified (FIG. 1 and Table 1).

TABLE 16 human pituitary hPRL protein forms and their distribution ratios among 5 pituitary tumor subtypes

The six human pituitary hPRL proteins exist in different forms and their distribution ratios among 5 pituitary tumor subtypes (table 1): compared with a control group (normal pituitary tissue), the non-functional pituitary tumor (NF-NFPA) without hormone expression has the advantages that the existence form v1 of hPRL protein is down-regulated by 8.3 times, v2 is down-regulated by 4.9 times, v3 is unchanged, and v4, v5 and v6 are deleted. In luteinizing hormone positive non-functional pituitary tumors (LH-NFPA), hPRL was downregulated 12.6 fold, 4.1 fold, 26.2 fold, 20.1 fold, 36.7 fold and 33.6 fold, respectively, in v1, v2, v3, v4, v5, v 6. In a non-functional pituitary tumor positive for follicle-hormone (FSH-NFPA), there was no change in v2 for hPRL, a loss of v5, a 46.2-fold drop in v1, a 14.6-fold drop in v3, a 17.6-fold drop in v4, and an 11.3-fold drop in v 6. FSH/LH positive non-functional pituitary tumors (FSH/LH-NFPA), v1, v2, v3, v4, v5, v6 of hPRL were down-regulated by 99.9, 3.8, 12.3, 19, 19.7 and 32.6 fold, respectively. However, in prolactinoma, compared to the control group (PRL: Con), hPRL was only 3.4-fold regulated at v1, whereas v2, v3, v4, v5, v6 were unchanged.

In addition, there were significant differences in the composition ratios of the 6 forms of hPRL protein presence between the 5 pituitary tumor subtypes (NF-, FSH +, LH +, FSH +/LH +, PRL + tumors), which were determined using the SPSS 22 software chi-square assay (p <0.05) (Table 2). At the same time, chi-square test was performed between each two pituitary tumor subtypes to find: except for FSH +/LH + and PRL +, the composition ratio of 6 hPRL protein existence forms between FSH + and PRL + has no significant difference (p >0.05), and any two pituitary tumor subtypes have significant difference (p < 0.05).

TABLE 2 Change in the constitutive ratio of the 6 forms of hPRL protein Presence between the five pituitary tumor subtypes

Note: comparative χ among 5 pituitary tumor subtypes²＝360.606，p＜0.01。

(II) verifying the form of hPRL protein by immunoaffinity imprinting based on two-dimensional electrophoresis

Western blot based dielectrophoresis combined with anti-hPRL antibodies and mass spectrometry are effective methods for verifying the presence of human pituitary hPRL proteins. Western blot was used to detect positive reactions in human pituitary tissue in the presence of four forms of hPRL protein, including v1, v4, v5 and v6 (FIG. 2, A is Western blot image and B is double gel silver stain image after protein conversion to PVDF membrane). Furthermore, the proteins on the 4 immunoblot positive spots are identified by MALDI-TOF-MS PMF and MALDI-TOF-TOF MS/MS and are all hPRL; the corresponding peptide fingerprint data are shown in table 3. Wherein v1 gives rise to two peptide fragments, including ions⁷²YTHGRGFITK⁸¹([M+H]⁺Mass to charge ratio of 1179.7) and¹¹⁸SWNEPLYHLVTEVR¹³¹([M+H]⁺quality ofThe charge ratio was 1743.0). v4 gave two identical peptide stretches as v 1. V6 also obtained in comparison with v1 and v4¹⁷¹ENEIYPVWSGLPSLQMADEESR¹⁹²([M+H]⁺Mass to charge ratio 2550.6); while MALDI-TOF-TOF MS/MS also analyzed the sample from v6¹¹⁸SWNEPLYHLVTEVR¹³¹The amino acid sequence of (a). Two peptide fragments¹¹⁸SWNEPLYHLVTEVR¹³¹([M+H]⁺Mass to charge ratio of 1743.0) and¹⁷¹ENEIYPVWSGLPSLQMADEESR¹⁹²([M+H]⁺mass to charge ratio 2550.2) were also identified at v 5.

TABLE 3 peptidic fingerprint data of tryptic peptide fragments

It can be seen that immunoblotting and mass spectrometry (FIG. 2) based on two-dimensional electrophoresis clearly verified the presence of hPRL protein in human pituitary tissue, v1, v4, v5, v6 (FIG. 1), but not v2 and v3 (FIG. 1); the possible factors are: the hPRL antibody is a common antibody and is not directed to a particular hPRL protein existing form variant. The relative molecular masses of v2 and v3 were greater than the normal hPRL relative molecular mass (25.9kDa), and it was evident that some adduct was added to v2 and v3, which may render it unreactive with hPRL antibodies. Although v2 and v3 were not validated by immunoblotting, v2 and v3 were clearly identified by mass spectrometry (table 1), which is a gold standard for identifying proteins. Thus, v2 and v3 are still considered to be forms of hPRL protein in human pituitary tissue.

(III) whether Signal peptide (1-28) is in the form of hPRL protein in human Pituitary tissue

The PRL in the human Swiss-Prot database contains 227 amino acids (1-227), with the signal peptide at amino acid positions 1-28. This is a precursor of hPRL, with a molecular weight of 25.9 kDa. Whereas mature hPRL has 199 amino acids (amino acid positions 29-227), a molecular weight of 22.9kDa and does not contain a signal peptide (amino acid positions 1-28). Therefore, it is necessary to determine whether the 6 identified forms of hPRL protein exist from the hormone precursor or the mature hPRL. Using PeptideMess Cleavage software, 7 characteristic tryptic peptides (1-4, 1-9, 1-38, 5-9, 5-38, 10-38, 10-44) were derived from hPRL precursors and 3 characteristic tryptic peptides (29-38, 29-44, 29-49) were derived from mature hPRL (Table 4). These characteristic tryptic peptide fragments can be used to identify whether the signal peptide (1-28) is contained in the form of hPRL protein. The PMF data and MS/MS data of all the hPRL proteins existing in the forms v1, v2, v3, v4, v5 and v6 are analyzed in a combined manner, and peptide fragments 29-38, 29-44 and 29-49 are not found (Table 4), so that the v1-v6 is not mature hPRL.

TABLE 4 tryptic peptides characteristic of the signal peptide (1-28) used to determine the presence of the hPRL protein

But a characteristic peptide segment MNIKGSPWK (positions 1-9, [ M + H ]]⁺Mass to charge ratio 1060.5608) (Table 4) was found in the mass spectrum of v1-v6, while the peptide segment MNIK (positions 1-4, [ M + H ]]⁺M/z 505.2803) and the peptide fragment GSPWK (positions 5-9, [ M + H ]]⁺m/z 574.2983) were not found because they were outside the mass spectrometric range; this indicates that 6 human pituitary tissue hPRL proteins exist in the form of a signal peptide.

From the 2DGE distribution pattern of the form in which the hPRL protein exists, it is known that the relative molecular masses of v1, v4, v5 and v6 are 26.0kDa,26.1kDa,25.9kDa and 25.9kDa, respectively, which are very close to the molecular mass of the hPRL precursor of 25.9 kDa. While the relative molecular masses of v2 and v3 were 26.4kDa and 27.9kDa, respectively, which were greater than the relative molecular mass of the hPRL precursor (25.9 kDa).

Therefore, from the mass spectral data and the 2DGE distribution pattern, the 6 hPRL proteins found in human pituitary tissue are all hPRL precursors rather than mature hPRL.

(IV) bioinformatics prediction of post-translational modifications on the form in which hPRL proteins exist

Selective cleavage and post-translational modification are two major factors contributing to the form of hPRL protein. By mass spectrometry and two-dimensional gel electrophoresis profiling of each hPRL protein form, none of the 6 hPRL protein forms identified were derived from cleavage. Thus, post-translational modifications may be a major factor in the form of the hPRL protein. On the 2DGE distribution pattern of the form of hPRL protein, v1, v4, v5 and v6 have very similar relative molecular masses but significantly different isoelectric points, which may be due to deamidation, resulting in a mass increase of one Da due to deamidation, with a much shifted isoelectric point. v2 and v3 have the same isoelectric point, different relative molecular masses (26.4kDa and 27.9kDa) which are larger than the hPRL precursor (25.9kDa), probably due to modification of hPRL by large chemical groups, such as glycosylation or phosphorylation. Glycosylation refers to a protein plus one or more covalently linked carbohydrates. Phosphorylation refers to the modification of proteins by free phosphate groups or complex 5' -phospho-DNA. The hPRL page of the UniProt database (https:// www.uniprot.org/unipret/P01236), page P01236, clearly indicates that hPRL (P01236) is a glycoprotein or phosphorylated protein.

Phosphorylation occurred at serine (S), threonine (T), and tyrosine (Y) residues, and the netphos3.1 server predicted significantly 14 pS sites, 5 pT sites, and 3 pY sites in hPRL.

The significant prediction of 10N-glycosylation sites in hPRL was made using NetNGlyc 1.0 Server.

A significant prediction of 6O-glycosylation sites in hPRL was made using NetOGlyc 4.0 Server.

Immunoblotting based on two-dimensional gel electrophoresis combined with anti-hPRL antibodies, mass spectrometry and bioinformatics were used to identify the presence of hPRL protein in human pituitary tissue and their differential expression profiles between different pituitary tumor subtypes. 6 forms of hPRL protein were identified in human pituitary tissue (v1, v2, v3, v4, v5, v 6). 6 hPRL proteins exist in 5 pituitary tumor subtypes (NF)^-,FSH⁺,LH⁺,FSH⁺/LH⁺,PRL⁺Tumors) are significantly different. Furthermore, 6 hPRL proteins are present in human pituitary tissue from hPRL precursors (1-227) of 227 amino acids in length with different post-translational modifications (e.g., deamidation, glycosylation and phosphorylation), rather than from hPRL mature bodies (29-227) of 199 amino acids in length. These findings provide new insights into the function and mechanism in human pituitary hPRL and its associated diseases, as well as the potential clinical value in pituitary tumors. The signal path involved in the existence form of hPRL protein can provide scientific basis for elucidating the biological functions of different existence forms of PRL protein and developing the medicine for blocking PRL signal path.

The above description is only for the preferred embodiment of the present invention, and is not intended to limit the present invention in any way. Any simple modification, change and equivalent changes of the above embodiments according to the technical essence of the invention are still within the protection scope of the technical solution of the invention.

Claims (2)

1. A method for identifying a biomarker for the presence of prolactin protein in human pituitary tissue, the method comprising:

s1, collecting pituitary adenoma and normal pituitary tissue samples, respectively cracking the tissues and extracting to respectively obtain pituitary adenoma tissue protein and normal pituitary tissue protein;

s2, dividing each group of the pituitary adenoma tissue protein obtained in the S1 and the normal pituitary tissue protein into two parts, respectively carrying out two-dimensional gel electrophoresis, carrying out the two-dimensional gel electrophoresis on the pituitary adenoma tissue protein to obtain a protein-containing two-dimensional gel a and a protein-containing two-dimensional gel A, and carrying out the two-dimensional gel electrophoresis on the normal pituitary tissue protein to obtain a protein-containing two-dimensional gel B and a protein-containing two-dimensional gel B;

s3, respectively carrying out western blotting on the protein-containing bidirectional gel a and the protein-containing bidirectional gel b obtained in S2, transferring the proteins on the bidirectional gels to polyvinylidene fluoride membranes to respectively obtain a PVDF membrane a and a PVDF membrane b, then the PVDF membrane a and the PVDF membrane b are respectively immersed in the sealing solution, shaken and sealed on a decoloring shaking table for 1 hour and then taken out, then soaking the mixture into primary antibody solution, shaking and incubating for 1h, washing and decolorizing with PBST solution for 4 times on a decolorizing shaker for 15min each time, washing with double distilled water for 2 times, then immersing the membrane into a second antibody solution, shaking and incubating for 1h, washing and decoloring for 4 times by using a PBST solution on a decoloring shaking table, washing and decoloring for 15min each time, and developing the treated PVDF membrane a and PVDF membrane b by using 5-bromo-4-chloro-3-indolyl phosphoric acid to obtain a visualized PVDF membrane a and a visualized PVDF membrane b;

the primary antibody solution is prepared by mixing a primary antibody solution and a secondary antibody solution in a volume ratio of 1: 1000 rabbit anti-human prolactin antibody and 0.3% bovine serum albumin-PBST solution;

the volume ratio of the secondary antibody solution is 1: 4000 parts of mixed solution of goat anti-rabbit alkaline phosphate IgG and bovine serum albumin-PBST solution with the mass fraction of 0.3%; the PBST solution is a phosphate buffer solution containing 0.2% of Tween 20 by mass;

s4, displaying the protein-containing bidirectional gel A and the protein-containing bidirectional gel B obtained in the S2 by a silver staining method to obtain a stained bidirectional gel A and a stained bidirectional gel B; displaying the protein-containing bidirectional gel a and the protein-containing bidirectional gel b subjected to western blotting in S3 by using a silver staining method to obtain a stained bidirectional gel a and a stained bidirectional gel b;

s5, scanning the visual PVDF film a and B obtained in S3, the dyed bidirectional gel A, the dyed bidirectional gel B, the dyed bidirectional gel a and the dyed bidirectional gel B obtained in S4 into digitized images, and introducing the digitized images into PDQuest^TMQuantifying the volume of protein spots in the software for analyzing the bidirectional gel image, and matching the protein spots which are positive in immunity with the protein spots of the corresponding dyed bidirectional gel a and the dyed bidirectional gel b;

s6, performing enzymolysis on proteins in the two-way gel protein spots corresponding to the western blot spots which are positive in immunity and are arranged in the visualized PVDF membrane a in S3 in the gel spots of the dyed two-way gel A and the dyed two-way gel a obtained in S4 by using trypsin, and extracting the trypsinEnzymatically digesting the peptide mixture with ZipTipC₁₈Purifying the microcolumn to obtain a purified trypsin enzymolysis peptide mixture a;

subjecting the gel spots of the stained bidirectional gel B and the stained bidirectional gel B obtained in S4, which correspond to the immunopositive western blot spots of the visualized PVDF membrane B obtained in S3, to trypsin enzymolysis, extracting a trypsin enzymolysis peptide mixture, and subjecting the trypsin enzymolysis peptide mixture to ZipTipC₁₈Purifying the microcolumn to obtain a purified trypsin enzymolysis peptide mixture b;

s7, carrying out MALDI-TOF-MS analysis on the purified trypsin enzymolysis peptide mixture a and the purified trypsin enzymolysis peptide mixture b obtained in S6 to obtain peptide fingerprint PMF data, carrying out LC-ESI-MS/MS analysis to obtain MS/MS data or carrying out MALDI-TOF-TOF-MS/MS analysis to obtain PMF data and MS/MS data, and obtaining original mass spectrum data;

s8, inquiring the original mass spectrum data obtained in S7 in a SwissProt human protein database, identifying human prolactin protein, comparing the original mass spectrum data obtained in each glue dot with a theoretical mass spectrum peak of the human prolactin protein, and identifying whether signal peptide is absent; predicting a prolactin phosphorylation site, an N-glycosylation site and an O-glycosylation site of human pituitary tissue by a NetPhos3.1 server, a NetNGlyc 1.0server and a NetOGlyc 4.0server respectively;

s9, analyzing the difference of the existence form ratio of the prolactin protein of the pituitary tissues among different pituitary tumor subtypes by using SPSS 22 software chi-square test.

2. The method for identifying the biomarkers of the presence of prolactin protein in human pituitary tissue according to claim 1, wherein the blocking solution in S3 is a mixture of 0.3% by weight bovine serum albumin-PBST solution and 0.1% by weight sodium azide-PBST solution at a volume ratio of 1: 1.

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