The relative influence of phosphorylation and methylation on responsiveness of peptides to MALDI and ESI mass spectrometry

Abstract

Qualitative and quantitative analysis of post-translational protein modifications by mass spectrometry is often hampered by changes in the ionization/detection efficiencies caused by amino acid modifications. This paper reports a comprehensive study of the influence of phosphorylation and methylation on the responsiveness of peptides to matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry. Using well-characterized synthetic peptide mixtures consisting of modified peptides and their unmodified analogs, relative ionization/detection efficiencies of phosphorylated, monomethylated, and dimethylated peptides were determined. Our results clearly confirm that the ion yields are generally lower and the signal intensities are reduced with phosphopeptides than with their nonphosphorylated analogs and that this has to be taken into account in MALDI and ESI mass spectrometry. However, the average reduction of ion yield caused by phosphorylation is more pronounced with MALDI than with ESI. The unpredictable impact of phosphorylation does not depend on the hydrophobicity and net charge of the peptide, indicating that reliable quantification of phosphorylation by mass spectrometry requires the use of internal standards. In contrast to phosphorylation, mono- and dimethylated peptides frequently exhibit increased signal intensities in MALDI mass spectrometry (MALDI-MS). Despite minor matrix-dependent variability, MALDI methods are well suited for the sensitive detection of dimethylated arginine and lysine peptides. Mono- and dimethylation of the arginine guanidino group did not significantly influence the ionization efficiency of peptides in ESI-MS.

Global dynamics of Escherichia coli phosphoproteome in central carbon metabolism under changing culture conditions

“…ACCEPTED MANUSCRIPT 13 we did not observe the general suppression of the phosphopeptide signal reported previously for ESI [51] .…”

“…Orbitrap instruments are able to use higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD) for fragmentation while the QSATR can only do collision induced dissociation (CID). However, considering the modest discovery rate for the unphosphorylated cognates, the data does not support the notion of phosphopeptides being inherently more difficult to detect by ESI-MS/MS [51] than by Orbitrap. This is further supported by the fact that the lower limit of detection (LLD) is similar for α-casein phosphopeptides and their unphosphorylated cognates (Fig.…”

“…It is often difficult to correctly assign the phosphor site and even when assignment is possible, many discovered sites are likely to be spurious signals due to non-specific kinase activity and phosphor-transfer during mass spectrometry [51]. For example, of the 105 phosphor sites originally reported by Macek and colleagues for E. coli growing in Luria-Bertani (LB) [15], only nine were found in their subsequent SILAC-based temporal study in glucose minimal M9 medium, while 141 new phosphor sites were reported [12].…”

“…by the negative charge suppression of phosphate groups, makes detection of phosphopeptides by ESI challenging [51]. Here we often found the proteins previously reported to be phosphorylated but the specific phosphorylated peptide was rarely detected.…”

SSPaQ: A Subtractive Segmentation Approach for the Exhaustive Parallel Quantification of the Extent of Protein Modification at Every Possible Site

“…A number of studies have used the relative signal intensities of the modified peptide and its corresponding nonmodified form to infer a degree of modification. However, since a modification can alter the ionization efficiency of a peptide to an unpredictable degree [7], the extent of modification of a peptide cannot reliably be determined by a simple comparison of these signal intensities.…”