Dataset Information

Over 10,000 peptide identifications from the HeLa proteome by using single-shot capillary zone electrophoresis combined with tandem mass spectrometry.

ABSTRACT: Capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) has recently attracted attention as a tool for shotgun proteomics. However, its performance for this analysis has so far fallen far below that of reversed-phase liquid chromatography (RPLC)-MS/MS. The use of a CZE method with a wide separation window (up to 90?min) and high peak capacity (ca. 300) is reported. This method was coupled to an Orbitrap Fusion mass spectrometer through an electrokinetically pumped sheath-flow interface for the analysis of complex proteome digests. Single-shot CZE-MS/MS lead to the identification of over 10?000 peptides and 2100 proteins from a HeLa cell proteome digest in approximately 100?min. This performance is nearly an order of magnitude better than earlier CZE studies and is within a factor of two to four of the state-of-the-art nano ultrahigh-pressure LC system.

SUBMITTER: Sun L

PROVIDER: S-EPMC4392885 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Over 10,000 peptide identifications from the HeLa proteome by using single-shot capillary zone electrophoresis combined with tandem mass spectrometry.

Sun Liangliang L Hebert Alexander S AS Yan Xiaojing X Zhao Yimeng Y Westphall Michael S MS Rush Matthew J P MJ Zhu Guijie G Champion Matthew M MM Coon Joshua J JJ Dovichi Norman J NJ

Angewandte Chemie (International ed. in English) 20141024 50

Capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) has recently attracted attention as a tool for shotgun proteomics. However, its performance for this analysis has so far fallen far below that of reversed-phase liquid chromatography (RPLC)-MS/MS. The use of a CZE method with a wide separation window (up to 90 min) and high peak capacity (ca. 300) is reported. This method was coupled to an Orbitrap Fusion mass spectrometer through an electrokinetically pumped sheath-flow inter ...[more]

PMID: 25346227

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Project description:Out of all categories, monoclonal antibody (mAb) therapeutics attract the most interest due to their strong therapeutic potency and specificity. Six of the 10 top-selling drugs are antibody-based therapeutics that will lose patent protection soon. The European Medicines Agency has pioneered the regulatory framework for approval of biosimilar products and approved the first biosimilar antibodies by the end of 2013. As highly complex glycoproteins with a wide range of micro-variants, mAbs require extensive characterization through multiple analytical methods for structure assessment rendering manufacturing control and biosimilarity studies particularly product and time-consuming. Here, capillary zone electrophoresis coupled to mass spectrometry by a sheathless interface (CESI-MS) was used to characterize marketed reference mAbs and their respective biosimilar candidate simultaneously over different facets of their primary structure. CESI-MS/MS data were compared between approved mAbs and their biosimilar candidates to prove/disconfirm biosimilarity regarding recent regulation directives. Using only a single sample injection of 200 fmol, CESI-MS/MS data enabled 100% amino acids (AA) sequence characterization, which allows a difference of even one AA between 2 samples to be distinguished precisely. Simultaneously glycoforms were characterized regarding their structures and position through fragmentation spectra and glycoforms semiquantitative analysis was established, showing the capacity of the developed methodology to detect up to 16 different glycans. Other posttranslational modifications hotspots were characterized while their relative occurrence levels were estimated and compared to biosimilars. These results proved the value of using CESI-MS because the separation selectivity and ionization efficiency provided by the system allowed substantial improvement in the characterization workflow robustness and accuracy. Biosimilarity assessment could be performed routinely with a single injection of each candidate enabling improvements in the biosimilar development pipeline.

Dataset Information

Over 10,000 peptide identifications from the HeLa proteome by using single-shot capillary zone electrophoresis combined with tandem mass spectrometry.

Publications

Over 10,000 peptide identifications from the HeLa proteome by using single-shot capillary zone electrophoresis combined with tandem mass spectrometry.

Similar Datasets

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