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Article

Top-Down Proteomics on a Chromatographic Time Scale Using Linear Ion Trap Fourier Transform Hybrid Mass Spectrometers

Supporting Info

Bryan A. Parks,^† Lihua Jiang,^† Paul M. Thomas,^† Craig D. Wenger,^† Michael J. Roth,^† Michael T. Boyne II,^† Patricia V. Burke,^‡ Kurt E. Kwast,^‡ and Neil L. Kelleher*^†

Department of Chemistry and Department of Molecular & Integrative Physiology and the Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Anal. Chem., 2007, 79 (21), pp 7984–7991

DOI: 10.1021/ac070553t

Publication Date (Web): October 5, 2007

†

Department of Chemistry.

‡

Department of Molecular & Integrative Physiology.

Corresponding author. E-mail: kelleher@scs.uiuc.edu.

Abstract

Proteomics has grown significantly with the aid of new technologies that consistently are becoming more streamlined. While processing of proteins from a whole cell lysate is typically done in a bottom-up fashion utilizing MS/MS of peptides from enzymatically digested proteins, top-down proteomics is becoming a viable alternative that until recently has been limited largely to offline analysis by tandem mass spectrometry. Here we describe a method for high-resolution tandem mass spectrometery of intact proteins on a chromatographic time scale. In a single liquid chromatography−tandem mass spectrometry (LC−MS/MS) run, we have identified 22 yeast proteins with molecular weights from 14 to 35 kDa. Using anion exchange chromatography to fractionate a whole cell lysate before online LC−MS/MS, we have detected 231 metabolically labeled (¹⁴N/¹⁵N) protein pairs from Saccharomyces cerevisiae. Thirty-nine additional proteins were identified and characterized from LC−MS/MS of selected anion exchange fractions. Automated localization of multiple acetylations on Histone H4 was also accomplished on an LC time scale from a complex protein mixture. To our knowledge, this is the first demonstration of top-down proteomics (i.e., many identifications) on linear ion trap Fourier transform (LTQ FT) systems using high-resolution MS/MS data obtained on a chromatographic time scale.

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