JP2015165208A - Method for analyzing glycoprotein using mass spectrometry - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve the analysis sensitivity for a glycopeptide and enhance the accuracy of the structural analysis for the glycopeptide by removing, with simple treatment, a peptide from a mixture of a glycopeptide obtained by performing enzyme digestion on a glycoprotein, with a peptide.SOLUTION: An acid decomposable surfactant is added as a protein solubilizer to a glycoprotein aqueous solution which is an analysis object (S1). Treatments such as denaturation, reductive alkylation, and trypsin digestion are conducted (S2 to S4). The acid decomposable surfactant in the solution has a hydrophobic portion and a hydrophilic portion, and a peptide and a glycopeptide generated by enzyme digestion bond with the portions, respectively due to the affinity thereof. Then, an acid is added, and the solution is left standing still for a predetermined time (S5) so that the bonds at the hydrophobic portion and the hydrophilic portion break. The hydrophobic portion which gets to the peptide due to the affinity precipitates. The precipitate is removed, and a sample is prepared from a supernatant mainly containing the glycopeptide to use it for mass spectrometry (S6 to S8).

Description

  The present invention relates to an analysis method for identifying a protein subjected to sugar chain modification, that is, a glycoprotein, or analyzing its structure using mass spectrometry.

  When performing identification or structural analysis of a protein using mass spectrometry, generally, the target protein is denatured and subjected to reductive alkylation treatment, and then enzyme digestion treatment with a protease such as trypsin is performed. Through a series of such pretreatments, a peptide fragment mixture is prepared by cleaving a part of peptide bonds of the protein. For example, a sample for matrix-assisted laser desorption / ionization (MALDI) is prepared from the peptide fragment mixture, and the sample is measured by a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOFMS) or the like.

   Since many proteins have high hydrophobicity, it is necessary to sufficiently dissolve the protein in a solvent using a solubilizing agent before performing the above-described series of pretreatments. However, it must be avoided that the solubilizing agent inhibits the protease activity during the digestion process or lowers the sensitivity in mass spectrometry. Therefore, it is necessary to select an appropriate compound as a solubilizing agent, and to perform an appropriate treatment to suppress such an influence when there is a possibility of adverse effects due to the solubilizing agent. For example, urea has excellent solubilizing properties, but may reduce enzyme activity. Therefore, when urea is used as a solubilizing agent, before the enzyme digestion reaction, urea is removed by ultrafiltration, or by reducing the urea concentration by dilution, the enzyme activity is increased and mass spectrometry is performed. It is often done to minimize the impact on.

  In recent years, solubilizers that can suppress the influence on mass spectrometry with a simpler treatment are also commercially available. For example, a compound called “RapiGest SF” (hereinafter, simply referred to as “rapigest”) manufactured by Waters, described in Non-Patent Documents 1 and 2, is one of them. FIG. 2 is a diagram showing the chemical structure of Lapigest and its acid reaction. Lapigest is an amphiphilic compound in which a hydrophobic part and a hydrophilic part are combined, and an acid-decomposable surfactant (ALS = Acid-) in which the bond between the hydrophobic part and the hydrophilic part is cleaved by an acid. Labile Surfactant). The official compound name of Lapigest is Sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl) methoxy] -1-propanesulfonate.

  Lapigest does not inhibit enzyme activity and does not modify proteins. In addition, when the aqueous solution of Lapigest is treated with an acid, the hydrophobic portion and the hydrophilic portion are disconnected and the hydrophobic portion is precipitated, so that the hydrophobic portion can be easily removed by centrifugation or the like. On the other hand, the hydrophilic portion can be easily removed by a reverse phase column or the like. Thereby, a sample substantially free of solubilizer can be prepared, and the influence on mass spectrometry can be suppressed.

  By the way, it is said that more than half of proteins constituting a living body are subjected to sugar chain modification, and the sugar chain modification plays an important role in the regulation of protein structure and function. Recent research has also revealed the relationship between various diseases such as immune diseases and abnormal sugar chain structures and abnormal glycation. For these reasons, structural analysis of glycoproteins has become very important in various fields such as life science, medicine, and drug development.

  In the case of mass spectrometry of glycoproteins, it has been common in the past to analyze the protein and the sugar chain part separately for the convenience of analysis and the like. However, with such a technique, it is difficult to specify the binding position of the sugar chain in the protein. Therefore, recently, a glycopeptide is produced by enzymatic digestion of only the protein portion without cleaving the sugar chain, and a structural analysis including the binding position of the sugar chain is performed by mass analysis of the mixture of the glycopeptide. Is increasing. In that case, the sample obtained by enzymatic digestion is a mixture of a glycopeptide and a peptide not subjected to glycosylation. When mass analysis is performed on this sample, an ion peak derived from the glycopeptide and an ion derived from the peptide are obtained. A mass spectrum with mixed peaks can be obtained.

  However, in general, since peptides that have not undergone post-translational modification are easily ionized compared to glycopeptides, the signal intensity of ions derived from glycopeptides is relatively low compared to the signal intensity of ions derived from peptides, In some cases, ions derived from glycopeptides may not be sufficiently observed. As a result, there is a problem that the accuracy of the structure analysis of the sugar chain portion and the identification of the binding position of the sugar chain is lower than the accuracy of the peptide structure analysis.

In order to avoid such a problem, conventionally, in many cases, one of the following processes is performed in the pre-process.
(1) After enzymatic digestion, the glycopeptide is roughly purified using sepharose, cellulose, graphite carbon, or the like. Thereby, since the ratio of the amount of the glycopeptide in the sample increases, the signal intensity of ions derived from the glycopeptide can be relatively increased.
(2) During enzymatic digestion, a plurality of types of proteases and pronases are used to finely cleave the peptide chain of the glycopeptide to produce a smaller glycopeptide. In general, a glycopeptide having a small molecular weight has higher detection sensitivity than a glycopeptide having a large molecular weight, and therefore, the signal intensity of ions derived from the glycopeptide can be relatively increased.

  All of the above methods are effective in relatively increasing the signal intensity of ions derived from glycopeptides, but the pretreatment work becomes complicated, which takes time and processing time, and reduces throughput. In addition, since the analysis target (glycoprotein, glycopeptide) is lost during the process, it is difficult to employ the above method when the amount of sample (the amount of protein in the sample) is small.

Go Masuda, Yasushi Ishibuchi, “Sample Preparation Method for Comprehensive Analysis of Membrane Proteins by Shotgun Proteomics,” Journal of the Mass Spectrometry Society of Japan (Journal of the Mass Spectrometry Society of Japan), Vol.57, No.3, 2009, pp.145-151 Ying Qing Yu, Martin Gilar, "Rapidest SF Surfactant: RAPIGEST SF SURFACTANT: AN ENABLING TOOL FOR IN -SOLUTION ENZYMATIC PROTEIN DIGESTION) ", Waters, [December 12, 2013 search], Internet <URL: http://www.waters.com/webassets/cms/library/docs/720003102en.pdf > Daniel J. Simpson and three others, “Improved In-Gel Digestion Results and Work-Flow Through the Youth of a Mass Spectrometry Compatible Surfactant ( Improved in-gel digestion results and work-flow through the use of a mass spectrometry compatible surfactant), Promega, [December 12, 2013 search], Internet <URL: http: //www.promega .jp / ~ / media / Files / Resources / Posters / ps094.pdf> “Expedeon PPS SILENT SERFECTANT”, Expedeon, [December 12, 2013 search], Internet <URL: http://shop.expedeon.com / products / 18-Protein-Solubility / 129-PPS-Silent-Surfactant / ＞ Jeremy L. Norris and three others, “Nonacid cleavable detergents applied to MALDI mass spectrometry profiling of whole cells), Journal of Mass Spectrometry, 2005, Vol. 40, pp. 1319-1326

  The present invention has been made to solve the above-described problems, and the object of the present invention is to enable detection of glycopeptides that are generally difficult to ionize with high sensitivity while minimizing the effort of pretreatment. Thus, it is to provide a glycoprotein analysis method using mass spectrometry, which can improve the accuracy of glycoprotein identification and structural analysis.

The present invention made to solve the above problems is a method for analyzing glycoproteins using mass spectrometry,
a) A protein having a structure including a hydrophobic part, a hydrophilic part, and a binding part that binds the hydrophobic part and the hydrophilic part and is cleaved under a predetermined condition to the glycoprotein to be analyzed. A peptide fragmentation step for cleaving the peptide bond of the glycoprotein by carrying out an enzymatic digestion process in the state of mixing the solubilizer;
b) a binding cleavage step of cleaving the binding portion of the protein solubilizing agent by carrying out the treatment under the predetermined conditions on the solution subjected to the enzyme digestion treatment;
c) a preparation step of removing the precipitate after the binding portion has been cut and preparing a sample for mass spectrometry from the supernatant;
The sample prepared by the preparation step is subjected to mass spectrometry.

  In the glycoprotein analysis method according to the present invention, the glycoprotein to be analyzed is solubilized using a protein solubilizer that includes a hydrophobic portion, a hydrophilic portion, and a binding portion that connects both of them. The solution thus prepared is subjected to an enzyme digestion process in the peptide fragmentation step, but an appropriate process such as a reductive alkylation process may be performed as necessary before the enzyme digestion process. In the peptide fragmentation step, digestion with an enzyme such as trypsin is performed, and the peptide bond of the glycoprotein is cleaved to generate a mixture of the glycopeptide and the peptide. In the solution after the enzymatic digestion treatment, due to the interaction due to affinity, the peptide is in contact with the hydrophobic part of the protein solubilizing agent, and the glycopeptide, particularly a glycopeptide having a large sugar chain part is in contact with the hydrophilic part of the protein solubilizing agent. , Attract each.

In the bond cleavage step, the bond between the hydrophobic part and the hydrophilic part of the protein solubilizer in which the peptide and glycopeptide are present in the state as described above is cleaved. At this time, the attractive interaction between the hydrophobic part and the peptide and between the hydrophilic part and the glycopeptide is maintained as they are. Therefore, the peptide attracting the hydrophobic part precipitates together with the hydrophobic part, while the glycopeptide attracting the hydrophilic part remains dissolved in the solution. In the next preparation step, the peptide attracted to the precipitated hydrophobic part is removed, and the supernatant of the solution is collected to recover the glycopeptide attracted to the hydrophilic part contained in the supernatant. And a sample is prepared from the solution containing this glycopeptide. When MALDI mass spectrometry is performed as mass spectrometry, a sample is prepared by adding a matrix at this time.
As described above, in the method for analyzing glycoprotein according to the present invention, since a peptide can be removed and a sample containing a large amount of glycopeptide can be prepared, the ion derived from the glycopeptide can be obtained by subjecting the sample to mass spectrometry. The sensitivity is improved.

In the method for analyzing glycoprotein according to the present invention, as the protein solubilizer, an acid-decomposable surfactant that can be cleaved by a treatment with an acid can be used. Specifically, in addition to “RapiGest SF” described above, compounds such as “ProteaseMAX” and “PPS Silent” described later can be used as protein solubilizers.
By using such a protein solubilizer, the binding portion of the protein solubilizer can be cleaved by a simple treatment such as treatment with an acid.

  According to the glycoprotein analysis method using mass spectrometry according to the present invention, peptides can be removed by simple pretreatment, and glycopeptides can be detected with high sensitivity. In particular, since glycopeptides having a larger sugar chain portion are more likely to remain in a sample, mass information relating to complex glycopeptides can be obtained accurately, and the accuracy of glycoprotein identification and structural analysis is improved.

The flowchart which shows the process sequence of the method which is one Example of the analysis method of the glycoprotein which concerns on this invention. The figure which shows the chemical structure of RapiGest SF, and its acid reaction. The figure which shows the detailed information of the glycopeptide contained in the tryptic digest of the glycoprotein used for experiment. It is the actual mass spectrum obtained with respect to the sample obtained from the transferrin digest, (a) is the mass spectrum with respect to the sample which performed acid treatment and sediment removal, (b) is acid treatment and sediment removal. Mass spectrum for samples not performed. It is the mass spectrum of the actual low mass-to-charge ratio range (m / z 500-3500) obtained with respect to the sample obtained from fetuin digest, (a) is with respect to the sample which performed acid treatment and deposit removal Mass spectrum, (b) is a mass spectrum for a sample not subjected to acid treatment and sediment removal. It is the mass spectrum of the actual high mass charge ratio range (m / z 3500-8500) obtained with respect to the sample obtained from the fetuin digest, (a) is with respect to the sample which performed acid treatment and deposit removal Mass spectrum, (b) is a mass spectrum for a sample not subjected to acid treatment and sediment removal.

Hereinafter, an embodiment of a glycoprotein analysis method using mass spectrometry according to the present invention will be described in detail with reference to the accompanying drawings.
FIG. 1 is a flowchart showing a processing procedure in the analysis method of this embodiment. Hereinafter, the processing procedure shown in FIG. 1 will be described with reference to a specific experimental example.

  In the experiment, RapiGest SF commercially available from Waters was used as a protein solubilizer. As described above, this is a kind of acid-decomposable surfactant, and its chemical structural formula and oxidation reaction are as shown in FIG. Two types of glycoproteins to be analyzed were transferrin and fetuin. FIG. 3 is a diagram showing detailed information of glycopeptides contained in a tryptic digest of glycoprotein used in the experiment.

  For transferrin digests, two types of glycopeptides with mass / charge ratios of m / z 3683 and m / z 4722 are referred to as Tf-GP1 and Tf-GP2. For fetuin digests, three types of glycopeptides with mass to charge ratios of m / z 4604, m / z 6536, and m / z 8279 are referred to as Fet-GP1, Fet-GP2, and Fet-GP3. FIG. 3 also shows the chemical structure of each glycopeptide, but it is natural that this structure is unknown in advance when analyzing the structure of an unknown glycoprotein.

  In the analysis method of this example, 1% (w / v) RapiGest SF aqueous solution as a protein solubilizer is mixed with an aqueous solution (2 ug / uL) of a glycoprotein (transferrin or fetuin) to be analyzed, A 100 mM dithiothreitol (DTT) aqueous solution and 100 mM ammonium bicarbonate were mixed so that the final concentrations were 0.1% (w / v), 10 mM, and 10 mM, respectively (step S1). Thereafter, in order to denature the glycoprotein, the mixed solution was heated at a temperature of 56 ° C. for 45 minutes to reduce the disulfide bond (step S2).

  Subsequently, 150 mM iodoacetamide (IAA) was added to the mixed solution so as to have a final concentration of 15 mM, and carbamide methylation was performed by shaking in the dark at room temperature for 45 minutes (step S3). Further, trypsin solution (200 ng / uL trypsin, 10 mM ammonium bicarbonate) is added to this solution (trypsin: substrate protein = 1: 50, w / w), and left overnight at a temperature of 37 ° C. The enzyme digestion process was implemented by this (step S4). By this enzymatic digestion, part of the peptide bond in the protein part of the glycoprotein is cleaved, and a mixture of a glycopeptide having a shorter amino acid sequence length than the glycoprotein and a peptide not subjected to glycosylation is generated. . Due to the presence of rapids in the solution, mainly due to affinity interactions, peptides are hydrophilic in the hydrophobic part of the protein solubilizer, and glycopeptides, especially glycopeptides with a large sugar chain, are hydrophilic in the protein solubilizer. With each department.

  The digestion reaction was stopped by adding 10% trifluoroacetic acid (TFA) to the above solution to a final concentration of 0.1% (v / v). The acid reaction was promoted by standing for 1 hour (in the case of transferrin digested product, 6 hours in the case of fetuin digested product) (step S5). Due to the action of the added trifluoroacetic acid, the bond between the hydrophobic part and the hydrophilic part of Lapigest is cleaved, and during the standing period in a low-temperature environment, the hydrophobic part is precipitated together with the peptide attracting it. On the other hand, the glycopeptide attracting the hydrophilic part of Lapigest remains in the solution. As a result, the glycoprotein-derived peptide and glycopeptide to be analyzed are separated.

  Thereafter, the precipitate was removed from the solution by centrifugation, and the supernatant portion was recovered (step S6). The recovered solution contains a large amount of glycopeptide attracting the hydrophilic portion of Lapigest. A sample for MALDI was prepared using the solution thus collected as a sample solution (step S7). That is, a sample solution and a predetermined matrix were dropped onto a MALDI sample plate, mixed, and dried under predetermined conditions to form a sample on the sample plate. The sample thus prepared was subjected to MALDI mass spectrometry, and mass analysis was performed in the positive ion mode to collect data (step S8).

  In the experiment, a sample was prepared using general DHB (2,5-Dioxybenzoic Acid) as a matrix, and mass spectrometry was performed using an AXIMA-Resonance manufactured by Shimadzu Corporation as a MALDI mass spectrometer. FIG. 4 is an actual mass spectrum obtained for a sample obtained from a tryptic digest of transferrin. (A) is a mass spectrum for a sample subjected to acid treatment and sediment removal, and (b) is an acid spectrum. It is a mass spectrum with respect to the sample which does not process and precipitate removal.

  In the mass spectrum shown in FIG. 4 (a), the peaks of proton-added molecular ions of glycopeptides Tf-GP1 and Tf-GP2 (marked with *), and sialic acid (Sialic acid) from glycopeptides Tf-GP1 and Tf-GP2, respectively. An ion peak (mass-to-charge ratio difference per sialic acid: 291) is observed. On the other hand, in the mass spectrum shown in FIG. 4B, the ion peaks derived from the glycopeptides Tf-GP1 and Tf-GP2 are hardly observed, and m / z 2500-2700, around m / z 3900-4000. In particular, peptide-derived ion peaks are observed with high intensity. From this, the acid treatment and the removal of the precipitate described above sufficiently remove the peptide that has not been sugar chain-modified (or from which the sugar chain has been detached), thereby observing ions derived from the glycopeptide with sufficient signal intensity. You can see that it is possible.

  Figures 5 and 6 are measured mass spectra obtained for samples obtained from tryptic digests for fetuin. Among these, FIG. 5 is a mass spectrum for m / z 500-3500 which is a relatively low mass-to-charge ratio range, and FIG. 6 is a mass spectrum for m / z 3500-8500 which is a higher mass-to-charge ratio range. 5 and 6, (a) is a mass spectrum for a sample subjected to acid treatment and precipitate removal, and (b) is a mass spectrum for a sample not subjected to acid treatment and precipitate removal.

  As can be seen from FIG. 6, the protonated molecular ion of glycopeptide Fet-GP1 (m / z 4604) is observed with a high signal intensity regardless of the presence or absence of acid treatment and precipitate removal. On the other hand, the ion peak derived from Fet-GP2 (m / z 6536) is observed with a slightly higher intensity when acid treatment and precipitate removal are not performed. In addition, the difference is remarkable in the ion peak derived from Fet-GP3 (m / z 8279), and when acid treatment and precipitate removal are performed, including an ion peak from which some sugar chains are desorbed, Although a plurality of ion peaks derived from Fet-GP3 were observed, when the acid treatment and the precipitate removal were not performed, the ion peaks derived from Fet-GP3 were hardly observed. This shows that the effect of peptide removal by acid treatment and precipitate removal is particularly effective for glycopeptides with a high mass-to-charge ratio in which almost no relevant ions can be detected without such treatment.

  As described above, a sample containing a large amount of glycopeptides derived from the glycoprotein to be analyzed can be prepared by the pretreatment in steps S1 to S7. Therefore, in mass spectrometry in step S7, glycopeptides, in particular sugars, are prepared. Mass information of glycopeptides having a large chain portion can be obtained. Thus, the mass information obtained in this way is used for database search such as Mascot provided by Matrix Science, or de novo sequencing to identify glycopeptides and the structure of glycoproteins. Is estimated (step S9).

In the above examples, RapiGest SF was used as a protein solubilizing agent. It can be used as a solubilizer.
The acid-decomposable surfactant is not particularly limited as long as it is a surfactant in which a hydrophilic part and a hydrophobic part are bonded with a linker having a structure or bond that is acid-decomposable. Specifically, in addition to RapiGest (Sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl) methoxy] -1-propanesulfonate), ProteinMAX ( The official compound name is Sodium 3-((1- (furan-2-yl) undecyloxy) carbonylamino) propane-1-sulfonate) and PPS Silent described in Non-Patent Document 4 etc. (The official compound name is Sodium 3- (4- (1,1-bis (hexyloxy) ethyl) pyridinium-1-yl) propane-1-sulfonate) and the like can be used.

Further, as a protein solubilizer, in addition to an acid-decomposable surfactant, a surfactant that decomposes by reaction with fluorine ions, a surfactant that decomposes by irradiation with ultraviolet light, and the like can also be used. As the surfactant that decomposes by reaction with fluorine ions, specifically, a surfactant containing a silyl ester group as a linker between a hydrophilic part and a hydrophobic part, for example, described in Non-Patent Document 5, 2- (Dimethyloctylsilanyl) ethoxycarbonylmethyl] -trimethylammonium bromide, [2- (Dimethyloctylsilanyl) ethoxycarbonylmethyl] -trimethylphosphonium bromide, and the like. On the other hand, as the surfactant that is decomposed by irradiation with ultraviolet light, any surfactant may be used as long as the structure or bond that is cleaved by ultraviolet light is included in the linker between the hydrophilic part and the hydrophobic part. (E) -3- (2,4,6-Trihydroxyphenyl) acrylic acid octyl ester described in the above.
Of course, depending on the type of protein solubilizer used and the type of glycoprotein to be analyzed, the parameters (temperature, time, concentration of compound used, etc.) in the series of pretreatments described above are appropriately changed. It is natural.

  In the above embodiment, the prepared sample was subjected to mass spectrometry using a MALDI time-of-flight mass spectrometer. However, it is possible to use a mass spectrometer using other ionization methods generally used for measuring peptides and the like. Good. For example, a mass spectrometer using another laser desorption ionization method such as a surface assisted laser desorption ionization (SALDI) method can also be used.

  Furthermore, the above-described embodiment is merely an example of the present invention, and other than the above-described modifications, any modifications, corrections, additions, etc. within the scope of the present invention are included in the scope of the claims of the present application. It is natural.

Claims (3)

A method for analyzing glycoproteins using mass spectrometry,
a) A protein having a structure including a hydrophobic part, a hydrophilic part, and a binding part that binds the hydrophobic part and the hydrophilic part and is cleaved under a predetermined condition to the glycoprotein to be analyzed. A peptide fragmentation step for cleaving the peptide bond of the glycoprotein by carrying out an enzymatic digestion process in the state of mixing the solubilizer;
b) a binding cleavage step of cleaving the binding portion of the protein solubilizer by carrying out the treatment under the predetermined conditions for the solution digested with the enzyme;
c) a preparation step for removing the precipitate after the bond cleavage step and preparing a sample for mass spectrometry from the supernatant;
A method for analyzing glycoprotein using mass spectrometry, wherein the sample prepared by the preparation step is subjected to mass spectrometry. A method for analyzing glycoprotein using mass spectrometry according to claim 1,
The method for analyzing glycoprotein using mass spectrometry, wherein the protein solubilizer is an acid-decomposable surfactant whose bond is cleaved by treatment with an acid. A method for analyzing glycoprotein using mass spectrometry according to claim 2,
The mass spectrometry is mass spectrometry based on matrix-assisted laser desorption / ionization, and the preparation step includes adding a predetermined matrix to prepare a sample, and a method for analyzing glycoprotein using mass spectrometry.

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