Iodoacetamide‐alkylated methionine can mimic neutral loss of phosphoric acid from phosphopeptides as exemplified by nano‐electrospray ionization quadrupole time‐of‐flight parent ion scanning

Abstract

Formation of S-carbamidomethylmethionine (camMet) occurs as a side reaction during cysteine alkylation with iodoacetamide (IAA). In collision-induced dissociation, peptides with camMet show an abundant neutral loss of 2-(methylthio)acetamide (C3H7NOS = 105.025 Da) at moderate collision offset values which are similar to those optimal for loss of phosphoric acid (H3PO4 = 97.977 Da). Neutral loss analysis is used for spotting of phosphopeptides which contain phosphoserine (pSer) or phosphothreonine (pThr) residues. In the case where precursor ions cannot be accurately assigned in the survey spectrum (e.g. due to low ion abundance or signal overlap), the mass accuracy of a neutral loss tandem mass spectrometry (MS/MS) analysis depends on the precursor ion isolation window. For the charge states 2+, 3+ or 4+, a typical 3.5 Da precursor isolation window results in neutral loss windows of 7, 10.5 or 14 Da, respectively. Consequently, neutral loss of 105 Da from alkylated methionine residues can mimic the phosphoserine/phosphothreonine-specific neutral loss of 98 Da. In the evaluation of quadrupole time-of-flight (QTOF) parent ion scan data for neutral loss of H3PO4, this interference was frequently observed. It is illustrated in this study using the analysis of ovalbumin phosphorylation as an example. The +80 Da molecular weight shift connected with phosphorylation at serine or threonine may also be mimicked by carbamidomethylation of methionine through a combination with sodium adduction (+57 Da +22 Da = +79 Da). For highly sensitive neutral loss analysis of serine and threonine phosphorylation, careful data inspection is recommended if reduction and alkylation by IAA is employed.

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

“…Cation-exchange chromatography was also employed as a means to separate the phosphorylated isoforms and determine the order of sequential H1 phosphorylation [150]. Identification of phosphorylation on serine and threonine residues was facilitated by the presence of corresponding neutral loss of phosphoric acid (98 Da in positive ion mode and 80 Da in negative mode) and data-dependent MS/MS/MS experiments [151][152].…”

Characterization of the Glycosylation Site of Human PSA Prompted by Missense Mutation using LC–MS/MS

“…Hence, D115, D116, and D182 were reflected as potential glycosylation sites. Oxidation and carbamidomethylation of methionine 33 were set as a variable modification while carbamidomethylation of cysteine was set as a fixed modification. Alkylated methionine is formed because of the reaction with iodoacetamide and it creates a neutral loss of 2-(methylthio)acetamide (C3H7NOS, 105.025Da) in CID MS/MS.…”

“…Alkylated methionine is formed because of the reaction with iodoacetamide and it creates a neutral loss of 2-(methylthio)acetamide (C3H7NOS, 105.025Da) in CID MS/MS. 33 Thus, a neutral loss of 2-(methylthio)acetamide in carbamidomethylation of methionine was set up for searching peptides and scoring ions. Regarding the ETD data, HexNAc2Hex5, HexNAc2Hex6, HexNAc4Hex5NeuAc2, and HexNAc4Hex5Fuc1NeuAc2 were set as additional variable modifications to sequence glycopeptides with glycan intact.…”

Analytical methodologies for the detection and structural characterization of phosphorylated proteins

“…Care must be taken to properly block the SH groups of Cys residues before derivatizations. To this purpose, performic acid oxidation [68] is preferred over alkylation [69] because alkylated Cys/Met residues may also undergo ␤-elimination [70]. Other phosphopeptide enrichment strategies such as IMAC may be coupled successfully to derivatization methods [71].…”

“…Various reagents have been used to this purpose, sometimes facilitating concomitant phosphopeptides enrichment and subsequent MS analysis [68][69][70][71][72][73][74][75][76]. Fig.…”