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Harnessing the power of electrophoresis and chromatography: Offline coupling of reverse phase liquid chromatography-capillary zone electrophoresis-tandem mass spectrometry for peptide mapping for monoclonal antibodies

ABSTRACT: RPLC-MS/MS is the present workhorse for bottom-up proteomics-based characterization. However it suffers from limited peak capacity and challenges with respect to detection of small and hydrophilic peptides. CZE-MS/MS offers an orthogonal alternative to RPLC-MS/MS but has limited sensitivity due to low sample loading. In the present work, for the first time, offline coupling of an RPLC-CZE-MS/MS has been demonstrated for peptide mapping of a monoclonal antibody-based therapeutic product. The performance of this platform has been compared to the state-of-the-art RPLC-MS/MS and CZE-MS/MS approaches. Fifteen fractions were isolated from a mAb tryptic digest using RPLC, and each fraction was analyzed by CZE-ESI-MS/MS. Results indicate that not only the RPLC-CZE-MS/MS platform identify a larger number of distinct peptides (372) than the RPLC-MS/MS (219) and CZE-MS/MS (177) platforms, it also offered significantly superior sequence coverage (99.55% vs. 89.76% and 94.21% for the heavy chain and 98.6% vs. 92.06% and 95.79% for the light chain). Also, the distribution of amino acid residues with post translational modifications was 413 for RPLC-CZE-MS/MS, 150 for RPLC-MS/MS and 94 for CZE-MS/MS. In fact, the RPLC-CZE-MS/MS system performed better than the combined datasets from RPLC-MS/MS or CZE-MS/MS. Our study shows that the traditionally used one-dimensional (1D) RPLC-MS/MS and CZE-MS/MS-based platforms may not be enough for characterization of complex molecules such as monoclonal antibodies. The proposed approach should be an essential addition to the analytical toolkit for in-depth primary structural characterization of mAbs.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Anurag Singh Rathore

LAB HEAD: Anurag Singh Rathore

PROVIDER: PXD017316 | Pride | 2020-03-05

REPOSITORIES: Pride

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Harnessing the power of electrophoresis and chromatography: Offline coupling of reverse phase liquid chromatography-capillary zone electrophoresis-tandem mass spectrometry for peptide mapping for monoclonal antibodies.

Kumar Ramesh R Shah Rohan L RL Rathore Anurag S AS

Journal of chromatography. A 20200207

RPLC-MS/MS is the present workhorse for bottom-up proteomics-based characterization. However it suffers from limited peak capacity and challenges with respect to detection of small and hydrophilic peptides. CZE-MS/MS offers an orthogonal alternative to RPLC-MS/MS but has limited sensitivity due to low sample loading. In the present work, for the first time, offline coupling of an RPLC-CZE-MS/MS has been demonstrated for peptide mapping of a monoclonal antibody-based therapeutic product. The perf ...[more]

PMID: 32063275

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Project description:Two-dimensional (2D) liquid chromatography (LC)-tandem mass spectrometry (MS/MS) are typically employed for deep bottom-up proteomics, and the state-of-the-art 2D-LC-MS/MS has approached over 8,000 protein identifications (IDs) from mammalian cell lines or tissues in 1-3 days of mass spectrometer time. Capillary zone electrophoresis (CZE)-MS/MS has been suggested as an alternative to LC-MS/MS for bottom-up proteomics. CZE-MS/MS and LC-MS/MS are complementary in protein/peptide ID from complex proteome digests because CZE and LC are orthogonal for peptide separation. In addition, the migration time of peptides from CZE-MS can be predicted accurately, which is invaluable for evaluating the confidence of peptide ID from the database search and even guiding the database search. However, the number of protein IDs from complex proteomes using CZE-MS/MS is still much lower than the state of the art using 2D-LC-MS/MS. In this work, for the first time, we established a strong cation exchange (SCX)-reversed phase LC (RPLC)-CZE-MS/MS platform for deep bottom-up proteomics. The platform identified around 8,200 protein groups and 65,000 unique peptides from a mouse brain proteome digest in 70 hours. The data represents the largest bottom-up proteomics dataset using CZE-MS/MS and provides a valuable resource for further improving the tool for prediction of peptide migration time in CZE. The peak capacity of the orthogonal SCX-RPLC-CZE platform was estimated to be around 7,000. SCX-RPLC-CZE-MS/MS produced comparable numbers of protein and peptide IDs with 2D-LC-MS/MS (8,200 vs. 8,900 protein groups, 65,000 vs. 70,000 unique peptides) from the mouse brain proteome digest using comparable instrument time. This is the first time that CZE-MS/MS showed its capability to approach comparable performance to the state-of-the-art 2D-LC-MS/MS for deep proteomic sequencing. SCX-RPLC-CZE-MS/MS and 2D-LC-MS/MS showed good complementarity in protein and peptide IDs and combining those two methods improved the number of protein group and unique peptide IDs by nearly 10% and over 40%, respectively, compared with 2D-LC-MS/MS alone.

			Action	DRS
	CEMSMSrun01.wiff	Wiff
	CEMSMSrun02.wiff	Wiff
	CEMSMSrun12.xlsx	Xlsx
	Fraction01run01.wiff	Wiff
	Fraction01run02.wiff	Wiff