Resolving the composition of protein complexes using a MALDI LTQ Orbitrap

Abstract

Current biological studies have been advanced by the continuous development of robust, accurate, and sensitive mass spectrometric technologies. The MALDI LTQ Orbitrap is a new addition to the Orbitrap configurations, known for their high resolving power and accuracy. This configuration provides features inherent to the MALDI source, such as reduced spectra complexity, forgiveness to contaminants, and sample retention for follow-up analyses with targeted or hypothesis-driven questions. Here we investigate its performance for characterizing the composition of isolated protein complexes. To facilitate the assessment, we selected two well characterized complexes from Saccharomyces cerevisiae, Apl1 and Nup84. Manual and automatic MS and MS/MS analyses readily resolved their compositions, with increased confidence of protein identification compared with our previous reports using MALDI QqTOF and MALDI IT. CID fragmentation of singly-charged peptides provided sufficient information for conclusive identification of the isolated proteins. We then assessed the resolution, accuracy, and sensitivity provided by this instrument in the context of analyzing the isolated protein assemblies. Our analysis of complex mixtures of singly-charged ions up to m/z 4000 showed that (1) the resolving power, inversely proportional to the square root of m/z, had over four orders of magnitude dynamic range; (2) internal calibration led to improved accuracy, with an average absolute mass error of 0.5 ppm and a distribution centered at 0 ppm; and (3) subfemtomole sensitivity was achieved using both CHCA and DHB matrices. Additionally, our analyses of a synthetic phosphorylated peptide in mixtures showed subfemtomole level of detection using neutral loss scanning.

Fig. 1. Characterization of protein complexes using the MALDI LTQ Orbitrap

Isolation of endogenous levels of genomically-tagged Apl1-EGFP and Nup84-PrA from yeast. The main observed interacting partners are indicated and color coded according to known (red) and novel (black) associations. The colors in the Nup84 isolation illustrate the presence of multiple subcomplexes of the yeast nuclear pore.

Fig. 2. Representative MALDI LTQ Orbitrap MS of selected proteins co-isolated with Apl1

The MS shows a mixture of peptides corresponding to Apl1, Apl3, Pma1 and GFP, co-migrating at ~110 kDa. Examples of peptides from each protein are labeled in the spectrum. The panel insets show expanded MS regions containing Apl1 and Apl3 peptides, illustrating the ability of the orbitrap to resolve adjacent peaks.

Fig. 3. Representative MALDI LTQ Orbitrap CID spectra of [M + H]⁺ ions of peptides from proteins co-isolated with Apl1

The small panel insets illustrate the MS regions containing the peptides selected for fragmentation. The corresponding monoisotopic masses are indicated. Following CID, the generated fragment ions were detected at the linear trap level.

Fig. 4. The resolving power of MALDI-LTQ Orbitrap XL as a function of m/z and relative peak intensity

Resolution values measured from MS spectra of the isolated Nup84 samples were scattered against m/z. Results are shown for spectra acquired at resolution settings of 60,000 (panel A) and 30,000 (panel B). The colors of the data points indicate relative peak intensity. Each data set, containing approximately 26,000 measurements, was split according to the peak intensity into —Top 1/3, —Middle 1/3 and —Low 1/3 categories and plotted separately. The peak intensities were drawn to the same scale for all plots. The color bars illustrate the corresponding intensity ranges, while the histograms represent the distribution of the observed peaks. The parameters of the correlations between resolving power and m/z are summarized in Panel C.

Fig. 5. Characterization of the mass accuracy of the MALDI LTQ Orbitrap

Mass errors of 109 peptides were calculated from externally (dark blue points) or internally (light pink triangles) calibrated MS spectra of proteins co-isolated with Nup84. A) The absolute values of mass errors are plotted against the m/z of the peptides (left panel). The distribution of the real mass errors is shown (right panel). B) Summary of mass error statistics.