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Deep Intact Proteoform Characterization in Human Cell Lysate Using High-pH and Low-pH Reversed-Phase Liquid Chromatography

  • Dahang Yu
  • Zhe Wang
  • Kellye A. Cupp-Sutton
  • Xiaowen Liu
  • Si WuEmail author
Focus: Protein Post-translational Modifications: Research Article

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

Top-down proteomics Mass spectrometry Liquid chromatography RPLC Intact proteoforms 

Notes

Acknowledgements

We thank Dr. Anthony Burgett for providing the Hela cells. This work was partly supported by grants from NIH NIAID R01AI141625, NIAID CSGADP Pilot project (NIH 5U01AI101990-04, BRI no. FY15109843), NIH NIGMS R01GM118470, OCAST HR16-125, and OU FIP program.

Supplementary material

13361_2019_2315_MOESM1_ESM.xlsx (344 kb)
ESM 1 (XLSX 344 kb)
13361_2019_2315_MOESM2_ESM.pdf (1.4 mb)
ESM 2 (PDF 1467 kb)

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© American Society for Mass Spectrometry 2019

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistryUniversity of OklahomaNormanUSA
  2. 2.School of Informatics and ComputingIndiana University-Purdue University IndianapolisIndianapolisUSA

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