Deep Intact Proteoform Characterization in Human Cell Lysate Using High-pH and Low-pH Reversed-Phase Liquid Chromatography
- 21 November 2019
- journal article
- research article
- Published by American Chemical Society (ACS) in Journal of the American Society for Mass Spectrometry
- Vol. 30 (12), 2502-2513
- https://doi.org/10.1007/s13361-019-02315-2
Abstract
Post-translational modifications (PTMs) play critical roles in biological processes and have significant effects on the structures and dynamics of proteins. Top-down proteomics methods were developed for and applied to the study of intact proteins and their PTMs in human samples. However, the large dynamic range and complexity of human samples makes the study of human proteins challenging. To address these challenges, we developed a 2D pH RP/RPLC-MS/MS technique that fuses high-resolution separation and intact protein characterization to study the human proteins in HeLa cell lysate. Our results provide a deep coverage of soluble proteins in human cancer cells. Compared to 225 proteoforms from 124 proteins identified when 1D separation was used, 2778 proteoforms from 628 proteins were detected and characterized using our 2D separation method. Many proteoforms with critically functional PTMs including phosphorylation were characterized. Additionally, we present the first detection of intact human GcvH proteoforms with rare modifications such as octanoylation and lipoylation. Overall, the increase in the number of proteoforms identified using 2DLC separation is largely due to the reduction in sample complexity through improved separation resolution, which enables the detection of low-abundance PTM-modified proteoforms. We demonstrate here that 2D pH RP/RPLC is an effective technique to analyze complex protein samples using top-down proteomics.Keywords
Funding Information
- National Institute of Allergy and Infectious Diseases (R01AI141625)
- National Institute of General Medical Sciences (R01GM118470)
- University of Oklahoma (FIP)
- Oklahoma Center for the Advancement of Science and Technology (HR16-125)
This publication has 64 references indexed in Scilit:
- Mapping intact protein isoforms in discovery mode using top-down proteomicsNature, 2011
- Studies on the effect of column angle in figure-8 centrifugal counter-current chromatographyJournal of Chromatography A, 2011
- Reversed‐phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10A cellsProteomics, 2011
- Size-Sorting Combined with Improved Nanocapillary Liquid Chromatography−Mass Spectrometry for Identification of Intact Proteins up to 80 kDaAnalytical Chemistry, 2010
- Hypusine-containing protein eIF5A promotes translation elongationNature, 2009
- Mass spectrometry identifies and quantifies 74 unique histone H4 isoforms in differentiating human embryonic stem cellsProceedings of the National Academy of Sciences of the United States of America, 2008
- Phosphopeptide elution times in reversed-phase liquid chromatographyJournal of Chromatography A, 2007
- NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteinsNucleic Acids Research, 2007
- Ensembl 2007Nucleic Acids Research, 2006
- Histone chaperone Asf1 is required for histone H3 lysine 56 acetylation, a modification associated with S phase in mitosis and meiosisProceedings of the National Academy of Sciences of the United States of America, 2006