Generic placeholder image

Protein & Peptide Letters

Editor-in-Chief

ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

Research Article

Mimicking LysC Proteolysis by ‘Arginine Modification-cum-Trypsin Digestion’: Comparison of Bottom-up & Middle-down Proteomic Approaches by ESI Q-TOF MS

Author(s): P. Boomathi Pandeswari, R. Nagarjuna Chary, A.S. Kamalanathan, Sripadi Prabhakar and Varatharajan Sabareesh*

Volume 28, Issue 12, 2021

Published on: 14 December, 2021

Page: [1379 - 1390] Pages: 12

DOI: 10.2174/0929866528666210929163307

Price: $65

Abstract

Background: Middle-down (MD) proteomics is an emerging approach for reliable identification of post-translational modifications and isoforms, as this approach focuses on proteolytic peptides containing > 25-30 amino acid residues (a.a.r.), which are longer than typical tryptic peptides. Such longer peptides can be obtained by AspN, GluC, and LysC proteases. Additionally, some special proteases were developed specifically to effect MD approach, e.g., OmpT, Sap9, etc. However, these proteases are expensive. Herein we report a cost-effective strategy ‘arginine modification- cum trypsin digestion’, which can produce longer tryptic peptides resembling LysC peptides derived from proteins.

Objective: The aim of this study is to obtain proteolytic peptides that resemble LysC peptides by using 'trypsin', which is a less expensive protease.

Methods: This strategy is based on the simple principle that trypsin cannot act at the C-termini of those arginines in proteins, whose sidechain guanidine groups are modified by 1,2-cyclohexanedione or phenylglyoxal.

Results: As a proof of concept, we demonstrate this strategy on four models: β-casein (bovine), β- lactoglobulin (bovine), ovalbumin (chick) and transferrin (human), by electrospray ionization-mass spectrometry (ESI-MS) involving hybrid quadrupole time-of-flight. From the ESI-MS of these models, we obtained several arginine modified tryptic peptides, whose lengths are in the range of 30-60 a.a.r. The collision induced dissociation MS/MS characteristics of some of the arginine modified longer tryptic peptides are compared with the unmodified standard tryptic peptides.

Conclusion: The strategy demonstrated in this proof-of-concept study is not only useful to obtain longer tryptic peptides that mimic LysC proteolytic peptides, but also facilitates in enhancing the probability of missed cleavages by the trypsin. Hence, this method aids in evading the possibility of obtaining very short peptides that are <5-10 a.a.r. Therefore, this is indeed a cost-effective alternative/ substitute for LysC proteolysis and, in turn, for those MD proteomic studies that utilize LysC. Additionally, this methodology can be fruitful for mass spectrometry-based de novo protein and peptide sequencing.

Keywords: Proteomics, Middle-Down Proteomics, Mass Spectrometry, Electrospray Ionization, Arginine modification, Trypticpeptides, 1, 2-Cyclohexanedione, Phenylglyoxal, Tandem mass spectrometry, Collision Induced Dissociation.

Graphical Abstract

[1]
Kaltashov, I.A.; Bobst, C.E.; Abzalimov, R.R. Mass spectrometry-based methods to study protein architecture and dynamics. Protein Sci., 2013, 22(5), 530-544.
[http://dx.doi.org/10.1002/pro.2238] [PMID: 23436701]
[2]
Aebersold, R.; Mann, M. Mass spectrometry-based proteomics. Nature, 2003, 422(6928), 198-207.
[http://dx.doi.org/10.1038/nature01511] [PMID: 12634793]
[3]
Aebersold, R. A mass spectrometric journey into protein and proteome research. J. Am. Soc. Mass Spectrom., 2003, 14(7), 685-695.
[http://dx.doi.org/10.1016/S1044-0305(03)00289-7] [PMID: 12837590]
[4]
Lane, C.S. Mass spectrometry-based proteomics in the life sciences. Cell. Mol. Life Sci., 2005, 62(7-8), 848-869.
[http://dx.doi.org/10.1007/s00018-005-5006-6] [PMID: 15868409]
[5]
Gillet, L.C.; Leitner, A.; Aebersold, R. Mass spectrometry applied to bottom-up proteomics: entering the high-throughput era for hypothesis testing. Annu. Rev. Anal. Chem. (Palo Alto, Calif.), 2016, 9(1), 449-472.
[http://dx.doi.org/10.1146/annurev-anchem-071015-041535] [PMID: 27049628]
[6]
Domon, B.; Aebersold, R. Mass spectrometry and protein analysis. Science, 2006, 312(5771), 212-217.
[http://dx.doi.org/10.1126/science.1124619] [PMID: 16614208]
[7]
Roe, M.R.; Griffin, T.J. Gel-free mass spectrometry-based high throughput proteomics: tools for studying biological response of proteins and proteomes. Proteomics, 2006, 6(17), 4678-4687.
[http://dx.doi.org/10.1002/pmic.200500876] [PMID: 16888762]
[8]
Brodbelt, J.S. Ion activation methods for peptides and proteins. Anal. Chem., 2016, 88(1), 30-51.
[http://dx.doi.org/10.1021/acs.analchem.5b04563] [PMID: 26630359]
[9]
Guthals, A.; Bandeira, N. Peptide identification by tandem mass spectrometry with alternate fragmentation modes. Mol. Cell. Proteomics, 2012, 11(9), 550-557.
[http://dx.doi.org/10.1074/mcp.R112.018556] [PMID: 22595789]
[10]
Kolbowski, L.; Belsom, A.; Rappsilber, J. Ultraviolet photodissociation of tryptic peptide backbones at 213 nm. J. Am. Soc. Mass Spectrom., 2020, 31(6), 1282-1290.
[http://dx.doi.org/10.1021/jasms.0c00106] [PMID: 32352297]
[11]
Jones, A.W.; Cooper, H.J. Dissociation techniques in mass spectrometry-based proteomics. Analyst (Lond.), 2011, 136(17), 3419-3429.
[http://dx.doi.org/10.1039/c0an01011a] [PMID: 21698312]
[12]
Pandeswari, P.B.; Sabareesh, V. Middle-down approach: a choice to sequence and characterize proteins/proteomes by mass spectrometry. RSC Adv., 2019, 9(1), 313-344.
[http://dx.doi.org/10.1039/C8RA07200K]
[13]
Chen, B.; Lin, Z.; Zhu, Y.; Jin, Y.; Larson, E.; Xu, Q.; Ge, Y. Middle down multi-attribute analysis of antibody-drug conjugates with electron transfer dissociation. Anal. Chem, 2019, 91(18), 11661-11669.
[http://dx.doi.org/10.1021/acs.analchem.9b02194]
[14]
Shaw, J.B.; Liu, W.; Vasil Ev, Y.V.; Bracken, C.C.; Malhan, N.; Guthals, A.; Beckman, J.S.; Voinov, V.G. Vasil′ev, Y. V.; Bracken, C. C.; Malhan, N.; Guthals, A.; Voinov, V. G. Direct determination of antibody chain pairing by top-down and middle-down mass spectrometry using electron capture dissociation and ultraviolet photodissociation. Anal. Chem., 2020, 92(1), 766-773.
[http://dx.doi.org/10.1021/acs.analchem.9b03129] [PMID: 31769659]
[15]
Crowe, S.O.; Rana, A.S.J.B.; Deol, K.K.; Ge, Y.; Strieter, E.R. Ubiquitin chain enrichment middle-down mass spectrometry enables characterization of branched ubiquitin chains in cellulo. Anal. Chem., 2017, 89(8), 4428-4434.
[http://dx.doi.org/10.1021/acs.analchem.6b03675] [PMID: 28291339]
[16]
Khatri, K.; Pu, Y.; Klein, J.A.; Wei, J.; Costello, C.E.; Lin, C.; Zaia, J. Comparison of collisional and electron-based dissociation modes for middle-down analysis of multiply glycosylated peptides. J. Am. Soc. Mass Spectrom., 2018, 29(6), 1075-1085.
[http://dx.doi.org/10.1007/s13361-018-1909-y] [PMID: 29663256]
[17]
Garcia, B.A. What does the future hold for top down mass spectrometry? J. Am. Soc. Mass Spectrom., 2010, 21(2), 193-202.
[http://dx.doi.org/10.1016/j.jasms.2009.10.014] [PMID: 19942451]
[18]
Sidoli, S.; Garcia, B.A. Middle-down proteomics: a still unexploited resource for chromatin biology. Expert Rev. Proteomics, 2017, 14(7), 617-626.
[http://dx.doi.org/10.1080/14789450.2017.1345632] [PMID: 28649883]
[19]
Jin, Y.; Lin, Z.; Xu, Q.; Fu, C.; Zhang, Z.; Zhang, Q.; Pritts, W.A.; Ge, Y. Comprehensive characterization of monoclonal antibody by Fourier transform ion cyclotron resonance mass spectrometry. MAbs, 2019, 11(1), 106-115.
[http://dx.doi.org/10.1080/19420862.2018.1525253] [PMID: 30230956]
[20]
Janssen, K.A.; Coradin, M.; Lu, C.; Sidoli, S.; Garcia, B.A. Quantitation of single and combinatorial histone modifications by integrated chromatography of bottom-up peptides and middle-down polypeptide tails. J. Am. Soc. Mass Spectrom., 2019, 30(12), 2449-2459.
[http://dx.doi.org/10.1007/s13361-019-02303-6] [PMID: 31512222]
[21]
Jiang, T.; Hoover, M.E.; Holt, M.V.; Freitas, M.A.; Marshall, A.G.; Young, N.L. Middle-down characterization of the cell cycle dependence of histone H4 posttranslational modifications and proteoforms. Proteomics, 2018, 18(11), e1700442.
[http://dx.doi.org/10.1002/pmic.201700442] [PMID: 29667342]
[22]
Moradian, A.; Kalli, A.; Sweredoski, M.J.; Hess, S. The top-down, middle-down, and bottom-up mass spectrometry approaches for characterization of histone variants and their post-translational modifications. Proteomics, 2014, 14(4-5), 489-497.
[http://dx.doi.org/10.1002/pmic.201300256] [PMID: 24339419]
[23]
Sidoli, S. Middle-down MS is ready to answer complex questions in chromatin biology. Proteomics, 2018, 18(13), e1800131.
[http://dx.doi.org/10.1002/pmic.201800131] [PMID: 29745061]
[24]
Coradin, M.; Mendoza, M.R.; Sidoli, S.; Alpert, A.J.; Lu, C.; Garcia, B.A. Bullet points to evaluate the performance of the middle-down proteomics workflow for histone modification analysis. Methods, 2020, 184, 86-92.
[http://dx.doi.org/10.1016/j.ymeth.2020.01.013] [PMID: 32070774]
[25]
Wu, S.L.; Kim, J.; Hancock, W.S.; Karger, B. Extended range proteomic analysis (ERPA): a new and sensitive LC-MS platform for high sequence coverage of complex proteins with extensive post-translational modifications-comprehensive analysis of beta-casein and epidermal growth factor receptor (EGFR). J. Proteome Res., 2005, 4(4), 1155-1170.
[http://dx.doi.org/10.1021/pr050113n] [PMID: 16083266]
[26]
Laskay, U.A.; Srzentić, K.; Fornelli, L.; Upir, O.; Kozhinov, A.N.; Monod, M.; Tsybin, Y.O. Practical considerations for improving the productivity of mass spectrometry-based proteomics. Chimia (Aarau), 2013, 67(4), 244-249.
[http://dx.doi.org/10.2533/chimia.2013.244] [PMID: 23967698]
[27]
Laskay, Ü.A.; Lobas, A.A.; Srzentić, K.; Gorshkov, M.V.; Tsybin, Y.O. Proteome digestion specificity analysis for rational design of extended bottom-up and middle-down proteomics experiments. J. Proteome Res., 2013, 12(12), 5558-5569.
[http://dx.doi.org/10.1021/pr400522h] [PMID: 24171472]
[28]
Pan, J.; Zhang, S.; Chou, A.; Borchers, C.H. Higher-order structural interrogation of antibodies using middle-down hydrogen/deuterium exchange mass spectrometry. Chem. Sci. (Camb.), 2016, 7(2), 1480-1486.
[http://dx.doi.org/10.1039/C5SC03420E] [PMID: 29910905]
[29]
Schräder, C.U.; Ziemianowicz, D.S.; Merx, K.; Schriemer, D.C. Simultaneous proteoform analysis of histones H3 and H4 with a simplified middle-down proteomics method. Anal. Chem., 2018, 90(5), 3083-3090.
[http://dx.doi.org/10.1021/acs.analchem.7b03948] [PMID: 29405698]
[30]
Srzentić, K.; Zhurov, K.O.; Lobas, A.A.; Nikitin, G.; Fornelli, L.; Gorshkov, M.V.; Tsybin, Y.O. Chemical-mediated digestion: an alternative realm for middle-down proteomics? J. Proteome Res., 2018, 17(6), 2005-2016.
[http://dx.doi.org/10.1021/acs.jproteome.7b00834] [PMID: 29722266]
[31]
van der Burgt, Y.E.M.; Kilgour, D.P.A.; Tsybin, Y.O.; Srzentić, K.; Fornelli, L.; Beck, A.; Wuhrer, M.; Nicolardi, S. Structural analysis of monoclonal antibodies by ultrahigh resolution MALDI in-source decay FT-ICR mass spectrometry. Anal. Chem., 2019, 91(3), 2079-2085.
[http://dx.doi.org/10.1021/acs.analchem.8b04515] [PMID: 30571088]
[32]
Srzentić, K.; Fornelli, L.; Tsybin, Y.O.; Loo, J.A.; Seckler, H.; Agar, J.N.; Anderson, L.C.; Bai, D.L.; Beck, A.; Brodbelt, J.S.; van der Burgt, Y.E.M.; Chamot-Rooke, J.; Chatterjee, S.; Chen, Y.; Clarke, D.J.; Danis, P.O.; Diedrich, J.K.; D’Ippolito, R.A.; Dupré, M.; Gasilova, N.; Ge, Y.; Goo, Y.A.; Goodlett, D.R.; Greer, S.; Haselmann, K.F.; He, L.; Hendrickson, C.L.; Hinkle, J.D.; Holt, M.V.; Hughes, S.; Hunt, D.F.; Kelleher, N.L.; Kozhinov, A.N.; Lin, Z.; Malosse, C.; Marshall, A.G.; Menin, L.; Millikin, R.J.; Nagornov, K.O.; Nicolardi, S.; Paša-Tolić, L.; Pengelley, S.; Quebbemann, N.R.; Resemann, A.; Sandoval, W.; Sarin, R.; Schmitt, N.D.; Shabanowitz, J.; Shaw, J.B.; Shortreed, M.R.; Smith, L.M.; Sobott, F.; Suckau, D.; Toby, T.; Weisbrod, C.R.; Wildburger, N.C.; Yates, J.R., III; Yoon, S.H.; Young, N.L.; Zhou, M. Interlaboratory study for characterizing monoclonal antibodies by top-down and middle-down mass spectrometry. J. Am. Soc. Mass Spectrom., 2020, 31(9), 1783-1802.
[http://dx.doi.org/10.1021/jasms.0c00036] [PMID: 32812765]
[33]
Lundblad, R. Modification of Arginine. In: Chemical Reagents for Protein Modification, Fourth Edition; CRC Press: Boca Raton, FL, 2014; pp. 625-660.
[http://dx.doi.org/10.1201/b16867-16]
[34]
Takahashi, K. The reaction of phenylglyoxal with arginine residues in proteins. J. Biol. Chem., 1968, 243(23), 6171-6179.
[http://dx.doi.org/10.1016/S0021-9258(18)94475-3] [PMID: 5723461]
[35]
Patthy, L.; Smith, E.L. Reversible modification of arginine residues. Application to sequence studies by restriction of tryptic hydrolysis to lysine residues. J. Biol. Chem., 1975, 250(2), 557-564.
[PMID: 234432]
[36]
Pandeswari, P.B.; Sabareesh, V.; Vijayalakshmi, M.A. Insights into stoichiometry of arginine modification by phenylglyoxal and 1, 2- cyclohexanedione probed by LC-ESI-MS. J Protein Proteomics, 2016, 7(4), 323-347.
[37]
Tyanova, S.; Temu, T.; Carlson, A.; Sinitcyn, P.; Mann, M.; Cox, J. Visualization of LC-MS/MS proteomics data in MaxQuant. Proteomics, 2015, 15(8), 1453-1456.
[http://dx.doi.org/10.1002/pmic.201400449] [PMID: 25644178]
[38]
Tyanova, S.; Temu, T.; Cox, J. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat. Protoc., 2016, 11(12), 2301-2319.
[http://dx.doi.org/10.1038/nprot.2016.136] [PMID: 27809316]
[39]
Leitner, A.; Amon, S.; Rizzi, A.; Lindner, W. Use of the arginine-specific butanedione/phenylboronic acid tag for analysis of peptides and protein digests using matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun. Mass Spectrom., 2007, 21(7), 1321-1330.
[http://dx.doi.org/10.1002/rcm.2967] [PMID: 17340573]
[40]
Brock, J.W.C.; Cotham, W.E.; Thorpe, S.R.; Baynes, J.W.; Ames, J.M. Detection and identification of arginine modifications on methylglyoxal-modified ribonuclease by mass spectrometric analysis. J. Mass Spectrom., 2007, 42(1), 89-100.
[http://dx.doi.org/10.1002/jms.1144] [PMID: 17143934]
[41]
Leitner, A.; Lindner, W. Effects of an arginine-selective tagging procedure on the fragmentation behavior of peptides studied by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Anal. Chim. Acta, 2005, 528(2), 165-173.
[http://dx.doi.org/10.1016/j.aca.2004.09.067]
[42]
Wanigasekara, M.S.K.; Chowdhury, S.M. Evaluation of chemical labeling methods for identifying functional arginine residues of proteins by mass spectrometry. Anal. Chim. Acta, 2016, 935, 197-206.
[http://dx.doi.org/10.1016/j.aca.2016.06.051] [PMID: 27543028]
[43]
Wanigasekara, M.S.K.; Huang, X.; Chakrabarty, J.K.; Bugarin, A.; Chowdhury, S.M. Arginine-selective chemical labeling approach for identification and enrichment of reactive arginine residues in proteins. ACS Omega, 2018, 3(10), 14229-14235.
[http://dx.doi.org/10.1021/acsomega.8b01729]
[44]
Pandeswari, P.B.; Sabareesh, V. An ESI Q-TOF study to understand the impact of arginine on CID MS/MS characteristics of polypeptides. Int. J. Mass Spectrom., 2021, 459, 116453.
[http://dx.doi.org/10.1016/j.ijms.2020.116453]

Rights & Permissions Print Cite
© 2024 Bentham Science Publishers | Privacy Policy