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

• Published in: Analytical chemistry (Washington) Vol. 79; no. 21; pp. 7984 - 7991
• Main Authors: Parks, Bryan A, Jiang, Lihua, Thomas, Paul M, Wenger, Craig D, Roth, Michael J, Boyne, Michael T, Burke, Patricia V, Kwast, Kurt E, Kelleher, Neil L
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.11.2007
• Subjects: Analytical biochemistry: general aspects, technics, instrumentation; Analytical chemistry; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chemistry; Chromatographic methods and physical methods associated with chromatography; Chromatography, Liquid - methods; Exact sciences and technology; Fourier Analysis; Fourier transforms; Fundamental and applied biological sciences. Psychology; Ion chromatography; Mass spectrometry; Molecular Weight; Other chromatographic methods; Proteins; Proteomics; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins - analysis; Sensitivity and Specificity; Spectrometric and optical methods; Tandem Mass Spectrometry - methods; Time Factors; High resolution; Fourier transform spectroscopy; On line; Peptides; Liquid chromatography; Protein; Ion exchange chromatography; Automatic processing; Anion exchange; Ion trap; Mass spectrometry MS/MS; Proteomics; Acetylation; Molecular mass
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac070553t

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.