Top-down proteomics on a chromatographic time scale using linear ion trap fourier transform hybrid mass spectrometers

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.

Figure 1

Workflow for LC–MS data summarizing steps taken for automated (bottom left) and manual (bottom right) data processing of LC–MS/MS data.

Figure 2

(A) Broadband FT mass spectrum and (B) 11+ protein charge state pair acquired at 7 T. (C) Single scan CID MS/MS data and (D) graphical fragment map showing fully automated identification of yeast ubiquitin.

Figure 3

Identification of Acb1p (Acyl-CoA binding protein; YGR037C) where expression of the ¹⁵N-labeled component was undetectable. (A) Intact FT mass spectrum and (B) fragmentation data are also shown (7 T). (C) The graphical fragmentation map and database search results.

Figure 4

TIC from full scan ion trap spectra obtained on a 7 T LTQ FT. Although not identified, high MW protein pairs are shown up to 44.6 kDa. Molecular weights were calculated via manual deconvolution.

Figure 5

TIC and representative identifications from top-down mass spectral analysis of a Y-PER extract on a 12 T LTQ FT. Notice that even late in the run confident identifications could still be made (D). The anomaly at 100 min comes from loss of spray current.

Figure 6

Yeast histone H4 analyzed by single-scan LC–MS/MS using an LTQ FT operating at 12 T. The diacetylated species at 809 m/z was isolated (A,*) and fragmented to give the MS/MS spectrum (B), with acetylation localized to the N-terminus and Lys16 (C).