Modular mass spectrometric tool for analysis of composition and phosphorylation of protein complexes

Abstract

The combination of high accuracy, sensitivity and speed of single and multiple-stage mass spectrometric analyses enables the collection of comprehensive sets of data containing detailed information about complex biological samples. To achieve these properties, we combined two high-performance matrix-assisted laser desorption ionization mass analyzers in one modular mass spectrometric tool, and applied this tool for dissecting the composition and post-translational modifications of protein complexes. As an example of this approach, we here present studies of the Saccharomyces cerevisiae anaphase-promoting complexes (APC) and elucidation of phosphorylation sites on its components. In general, the modular concept we describe could be useful for assembling mass spectrometers operating with both matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) ion sources into powerful mass spectrometric tools for the comprehensive analysis of complex biological samples.

Figure 1

Schematic diagrams of (A) a tandem mass spectrometer, and (B) a modular mass spectrometer.

Figure 2

Combined performances of a prOTOF and a vMALDI-LTQ mass spectrometers as one modular tool. (A) prOTOF-MALDI-MS spectrum of a 1 femtomole mixture of six peptides, obtained in 30 seconds of spectrum acquisition time. The measurements of the m/z values of the peptides were performed using an external instrument calibration. The monoisotopic resolution for the detected ion peaks as well as the calculated (c) and the experimental (e) m/z values are shown. The peptides are bradykinin (fragment 2–9: PPGFSPFR, m/z = 904.468, theoretical value), Substance P (RPKPQQFFGLM-NH2, m/z = 1347.736), neurotensin (pELYENKPRRPYIL, m/z = 1672.918), amyloid β-protein (fragment 12–28: VHHQKLVFFAEDVGSNK, m/z = 1955.014), ACTH (SYSMEHFRWGKPVGKKRRPVKVYP, m/z = 2932.588), and B chain of oxidized insulin (FVNQHLC(O3)GSHLVEALYLVC(O3)GERGFFYTPKA, m/z = 3494.651). (B) vMALDI-LTQ MS/MS spectra of all 6 detected peptides. All spectra were measured in 2–3 seconds after the automatic collection of the MS/MS spectra. The interpretation of the observed fragmentation spectra and the identity of the observed peptides are indicated in each panel.

Figure 3

The flow of steps in the experiments for characterization of the subunits of the S. cerevisiae APC with a modular mass spectrometric tool.

Figure 4

(A) A map of neutral losses of 98 Da detected in all MS/MS spectra obtained from the sample shown in Fig. 3. The APC was purified from ∼(2–4)×10¹⁰ cells. (B) A map of neutral losses of 98 Da detected in all MS/MS spectra obtained from APC purified from a large culture of about 10¹¹ cells. All peaks with signal-to-noise ratios above 10-to-1 were examined by MS/MS and MS/MS/MS to confirm the loss of 98 Da and to identify the phosphopeptides and plausible position of a phosphate group. Peaks indicated with an asterisk were not identified. (C) Summary of the identified phosphopeptides from proteins co-immunopurified with the Cdc16-3xFlag-6xH protein.

Figure 5

(A) An MS/MS spectrum of a phosphopeptide detected at m/z 2789.245. The loss of 98 Da, which is a specific signature of phosphorylation, is indicated. (B) MS/MS/MS of the fragment that lost 98 Da. The identity of the phosphopeptide and the plausible location of the residue which lost the phosphate group are shown in the panel.