Chimera Spectrum Diagnostics for Peptides Using Two-Dimensional Partial Covariance Mass Spectrometry

Abstract

The rate of successful identification of peptide sequences by tandem mass spectrometry (MS/MS) is adversely affected by the common occurrence of co-isolation and co-fragmentation of two or more isobaric or isomeric parent ions. This results in so-called `chimera spectra', which feature peaks of the fragment ions from more than a single precursor ion. The totality of the fragment ion peaks in chimera spectra cannot be assigned to a single peptide sequence, which contradicts a fundamental assumption of the standard automated MS/MS spectra analysis tools, such as protein database search engines. This calls for a diagnostic method able to identify chimera spectra to single out the cases where this assumption is not valid. Here, we demonstrate that, within the recently developed two-dimensional partial covariance mass spectrometry (2D-PC-MS), it is possible to reliably identify chimera spectra directly from the two-dimensional fragment ion spectrum, irrespective of whether the co-isolated peptide ions are isobaric up to a finite mass accuracy or isomeric. We introduce '3-57 chimera tag' technique for chimera spectrum diagnostics based on 2D-PC-MS and perform numerical simulations to examine its efficiency. We experimentally demonstrate the detection of a mixture of two isomeric parent ions, even under conditions when one isomeric peptide is at one five-hundredth of the molar concentration of the second isomer.

Keywords: chimera spectra; tandem mass spectrometry; two-dimensional partial covariance mass spectrometry.

Figure 1

Three-dimensional view of the 2D-PC-MS maps of [GSNKGAIIGLM+2H]²⁺ (I1²⁺, top) and [MLGIIAGKNSG+2H]²⁺ (I2²⁺, bottom). The standard 1D tandem mass spectra are appended to the x- and y-axes for visual clarity. The single primary mass conservation line, characteristic of doubly charged peptides (see Equation (4)), is shown by the dashed line. Fragment-fragment correlations between the complementary b- and y-fragments lying on the primary mass conservation line are annotated.

Figure 2

2D-PC-MS map of the chimera spectrum of a mixture of [GSNKGAIIGLM + 2H]²⁺ (I1²⁺) and [MLGIIAGKNSG + 2H]²⁺ (I2²⁺) at a relative molar concentration of 1:499. The standard 1D tandem mass spectrum of the mixture is appended to the x- and y-axes for visual clarity. The autocorrelation diagonal of the 2D-PC-MS map, with respect to which it is symmetric, is shown by a full line. The autocorrelation signals of each and every fragment with itself are removed for clarity. The single primary mass conservation line, characteristic of doubly charged peptides (see Equation (4)), is shown by the blue dashed line. Fragment-fragment correlations are shown by circles, color-coded according to the values of their 2D-PC-MS correlation scores, Equation (3). 2D-PC-MS correlations of the complementary fragment ions are located on the primary mass conservation line. Two groups of three such correlations are found to lie within $\Delta$mass < 57 Da mass range, representing the two 3-57 chimera tags, shown by the vertical dotted lines. The identities of the complementary fragments forming the chimera tag correlations are also shown, although assignment of the complementary ions is not necessary for the 3-57 chimera tag identification; see text for details.

Figure 3

Histograms of the number of chimera tags predicted to be measured by 2D-PC-MS within the positive ion mode CID for two-, three-, and four-fold chimera spectra of 2+ (left) and 3+ (right) tryptic peptide ions derived from the Swiss-Prot database. The lowest rate of chimera spectra detection is predicted to be approximately 83% (in the two-fold chimera spectra of the doubly charged ions), demonstrating the usefulness of the approach.