Top‐down mass spectrometry of intact phosphorylated β‐casein: Correlation between the precursor charge state and internal fragments

Abstract

Phosphorylated proteins play essential roles in many cellular processes, and identification and characterization of the relevant phosphoproteins can help to understand underlying mechanisms. Herein, we report a collision‐induced dissociation top‐down approach for characterizing phosphoproteins on a quadrupole time‐of‐flight mass spectrometer. β‐casein, a protein with two major isoforms and five phosphorylatable serine residues, was used as a model. Peaks corresponding to intact β‐casein ions with charged states up to 36+ were detected. Tandem mass spectrometry was performed on β‐casein ions of different charge states (12+, and 15+ to 28+) in order to determine the effects of charge state on dissociation of this protein. Most of the abundant fragments corresponded to y, b ions, and internal fragments caused by cleavage of the N‐terminal amide bond adjacent to proline residues (Xxx‐Pro). The abundance of internal fragments increased with the charge state of the protein precursor ion; these internal fragments predominantly arose from one or two Xxx‐Pro cleavage events and were difficult to accurately assign. The presence of abundant sodium adducts of β‐casein further complicated the spectra. Our results suggest that when interpreting top‐down mass spectra of phosphoproteins and other proteins, researchers should consider the potential formation of internal fragments and sodium adducts for reliable characterization.

Internal Fragments Generated by Electron Ionization Dissociation Enhance Protein Top-Down Mass Spectrometry

“…Kelleher and co-workers showed that internal fragments from CID fragmentation of the common test protein, ubiquitin (8.6 kDa), can be assigned to result in significantly greater protein sequence coverage [21]. Similarly, for other intact proteins, the inclusion of internal fragments that can be generated by CID could result in greater explanation of the protein sequence [27,28]. Our laboratory demonstrated that internal product ions can be generated from top-down MS of large, native protein complexes [29].…”

“…20 Similarly, for other intact proteins, the inclusion of internal fragments that can be generated by CID could result in greater explanation of the protein sequence. 27,28 Our laboratory demonstrated that internal product ions can be generated from top-down MS of large, native protein complexes. 29 These examples suggest that the inclusion of internal fragments in top-down protein sequencing experiments could significantly enhance the protein sequence coverage and the efficiency of top-down mass spectrometry experiments.…”

Using Native Mass Spectrometry to Analyse Proteins Directly from Food

“…A key advantage to top down proteomic (TDP) methods, is the opportunity to localise PTMs within proteins. [93][94][95] This approach has recently been employed to identify the region of phosphorylation (between the 14 th and 50 th AA residue) for commercially obtained cow βcasein sprayed from denaturing solution conditions, 96 although this approach would also fit within our native MS workflow. 97 The fragmentation map highlights the fragments produced from the [M+Ca+Na+5H] 8+ ion subjected to 145V HCD CE.…”

Top-down mass spectrometry and assigning internal fragments for determining disulfide bond positions in proteins

“…53 While the analysis of internal fragment ions has been largely ignored by the TD-MS community due to the general lack of software tools to accurately and reliably assign them, the concept of the formation of internal fragment ions in TD-MS spectra is not novel. Previous studies have shown that the inclusion of internal fragments results in much richer sequence information of small peptides, 51,[54][55][56][57] intact proteins, [58][59][60][61][62][63][64] protein complexes, 65,66 and aid the identification of ambiguous proteoforms in mammalian cell lysates by topdown ptoteomics. 67 In addition, a recent study by Chin et al demonstrated the utility of internal fragments to enhance sequence coverage and to decipher disulfide bonds of disulfide-rich peptides.…”