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.
Highlights
Tandem mass spectrometry (MS/MS) has become the method of choice for the analysis of protein molecules, following major technological advancements in sample preparation, measurement hardware and data analysis techniques over the past several decades [1]
The recently introduced method of two-dimensional partial covariance mass spectrometry (2D-PC-MS) [9] allows unambiguous identification of the complementary ion pairs based on the geometrical position of their correlation signals on the 2D map, providing, as we show here, a direct route to diagnostics of the chimera spectra
Our numerical results show that the 2D-PC-MS chimera diagnostics based on the 3-57 tags introduced in this work is a reliable tool for identifying co-fragmentation of multiple isobaric or isomeric peptides
Summary
Tandem mass spectrometry (MS/MS) has become the method of choice for the analysis of protein molecules, following major technological advancements in sample preparation, measurement hardware and data analysis techniques over the past several decades [1]. One of the challenges still faced by the technique is the pervasive problem of so-called chimera spectra, where two or more parent ions are co-selected for fragmentation in the MS/MS workflow [2]. This is estimated to occur for up to ∼50% of all fragment ion spectra in a standard HPLC-MS run [2,3]. Chimera spectra hinder the automated sequence-to-spectrum assignment, for example, via reduction of the scores of the correct peptide sequences in the presence of contaminant fragment ions of a co-fragmented sequence [2].
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