Neutral Loss-Based Phosphopeptide Recognition:  A Collection of Caveats

Abstract

The standard strategy for analysis by tandem mass spectrometry of protein phosphorylation at serine or threonine utilizes the neutral loss of H3PO4 (= 97.977/z) from proteolytic peptide molecular ions as marker fragmentation. Manual control of automatically performed neutral loss-based phosphopeptide identifications is strongly recommended, since these data may contain false-positive results. These are connected to the experimental neutral loss m/z error, to competing peptide fragmentation pathways, to limitations in data interpretation software, and to the general growth of protein sequence databases. The fragmentation-related limitations of the neutral loss approach cover (i) the occurrence of abundant 'close-to-98/z' neutral loss fragmentations, (ii) the erroneous assignment of a neutral loss other than loss of H3PO4 due to charge state mix-up, and (iii) the accidental occurrence of any fragment ion in the m/z windows of interest in combination with a charge-state mix-up. The 'close-to-98/z' losses comprise loss of proline (97.053/z), valine (99.068/z), threonine (101.048/z), or cysteine (103.009/z) preferably from peptides with N-terminal sequences PP, VP, TP, or CP, and loss of 105.025/z from alkylated methionine. Confusion with other neutral losses may occur, when their m/z window coincides with a 98/z window as result of a charge state mix-up. Neutral loss of sulfenic acid from oxidized methionine originating from a doubly charged precursor (63.998/2 = 31.999) may thus mimic the loss of phosphoric acid from a triply charged phosphopeptide (97.977/3 = 32.659). As a consequence of the large complexity of proteomes, peptide sequence ions may occur in one of the mass windows of H3PO4 loss around 97.977/z. Practical examples for false-positive annotations of phosphopeptides are given for the first two groups of error. The majority of these can be readily recognized using the guidelines presented in this study.

Large-Scale Examination of Factors Influencing Phosphopeptide Neutral Loss during Collision Induced Dissociation

“…Loss of 98 Da in an MS 2 spectrum is a positive indicator of the presence of phosphorylation, barring a few caveats[8]. In practice, however, the degree of neutral loss is highly variable and in some cases not present at all, depending on the peptide sequence[9].…”

Tandem mass spectrometry strategies for phosphoproteome analysis

“…In particular, it has been noted that the neutral loss product ions are more abundant at higher collision energies (Schlosser et al, 2001;Hoffman et al, 2006), and in mass spectrometers that deposit internal energy within a short activation timescale (e.g., msec), such as in triple quadrupole instruments (Lehmann et al, 2007). The identity (i.e., structure) of the phosphorylated amino acid also affects the extent to which the various neutral loss product ions are observed by CID.…”

Facts and artifacts in proteomics of body fluids. What proteomics of saliva is telling us?

“…Further interpretative pitfalls derive, as mentioned before, from several neutral losses occurring during the fragmentation process. This problem has been recently outlined for neutral-loss based phosphopeptide recognition [74]. Detection of abundant "close-to-98/z" neutral loss fragmentations, erroneous assignment of H 3 PO 4 neutral losses, due to charge state mix-up, as well as accidental occurrence of any fragment ion in the m/z window of interest in combination with a charge-state mix-up, are often responsible for false-positive annotations.…”