Phosphopeptide Fragmentation and Site Localization by Mass Spectrometry: An Update

Potel, Clement M; Lemeer, Simone; Heck, Albert J. R.

doi:10.1021/acs.analchem.8b04746

Cited by 96 publications

(129 citation statements)

References 164 publications

Supporting

Mentioning

120

Contrasting

Unclassified

Order By: Relevance

“…As predicted, we observed a pTyr immonium ion at m/z 216.04, although this was present in fewer spectra than expected, being observed in 14.5% of confidently localised pTyr‐containing peptides ( ptm RS ≥ 0.99), decreasing to 10.7% at ptm RS ≥ 0.90. These findings are broadly in agreement with a previous report stating that this pTyr immonium ion was only observed in ~20% of HCD spectra from pTyr peptides when present in a mixture with other types of phosphopeptides, although the rationale for the decrease in pTyr immonium ions in extremely complex mixtures has yet to be explained (Potel et al , 2018a). Moreover, although the presence of an ion at m/z 216.04 is generally deemed indicative of the presence of pTyr, 5.0% (± 2.7% dependent on the nature of the phosphorylated residue) of HCD spectra from all other pX‐containing phosphopeptides also contained an ion at this m/z value.…”

Section: Resultssupporting

confidence: 93%

Strong anion exchange‐mediated phosphoproteomics reveals extensive human non‐canonical phosphorylation

et al. 2019

View full text Add to dashboard Cite

Phosphorylation is a key regulator of protein function under (patho)physiological conditions, and defining site‐specific phosphorylation is essential to understand basic and disease biology. In vertebrates, the investigative focus has primarily been on serine, threonine and tyrosine phosphorylation, but mounting evidence suggests that phosphorylation of other “non‐canonical” amino acids also regulates critical aspects of cell biology. However, standard methods of phosphoprotein characterisation are largely unsuitable for the analysis of non‐canonical phosphorylation due to their relative instability under acidic conditions and/or elevated temperature. Consequently, the complete landscape of phosphorylation remains unexplored. Here, we report an unbiased phosphopeptide enrichment strategy based on strong anion exchange (SAX) chromatography (UPAX), which permits identification of histidine (His), arginine (Arg), lysine (Lys), aspartate (Asp), glutamate (Glu) and cysteine (Cys) phosphorylation sites on human proteins by mass spectrometry‐based phosphoproteomics. Remarkably, under basal conditions, and having accounted for false site localisation probabilities, the number of unique non‐canonical phosphosites is approximately one‐third of the number of observed canonical phosphosites. Our resource reveals the previously unappreciated diversity of protein phosphorylation in human cells, and opens up avenues for high‐throughput exploration of non‐canonical phosphorylation in all organisms.

show abstract

Section: Resultssupporting

confidence: 93%

Strong anion exchange‐mediated phosphoproteomics reveals extensive human non‐canonical phosphorylation

et al. 2019

View full text Add to dashboard Cite

show abstract

“…Whereas phosphate group loss decreases the amount of information obtained, even more troubling is the possibility of phosphosite scrambling (Scheme C), which can lead to erroneous phosphosite localization. The observation that phosphopeptides can undergo an intramolecular transfer during in‐trap CID was most notably observed by Palumbo and Reid, and since then, Reid and others have attempted to determine how residue identity, activation energy, and activation timescale contribute to the degree of phosphate group rearrangement . On the one hand, some proteomics studies have suggested that phosphate group scrambling is merely a minor problem, minimally affecting sequencing efforts (<1% phosphopeptides producing incorrect sequences) .…”

Section: Introductionmentioning

confidence: 99%

“…The observation that phosphopeptides can undergo an intramolecular transfer during in-trap CID was most notably observed by Palumbo and Reid, 6 and since then, Reid and others have attempted to determine how residue identity, activation energy, and activation timescale contribute to the degree of phosphate group rearrangement. [7][8][9][10][11][12][13][14] On the one hand, some proteomics studies have suggested that phosphate group scrambling is merely a minor problem, minimally affecting sequencing efforts (<1% phosphopeptides producing incorrect sequences). 10 On the other hand, the phosphopeptide systems studied by Cui and Reid exhibited high transfer ratios (approximately 50 times greater than average), 12 which beg the question what mechanism may be at play, and how general this mechanism may be.…”

Section: Introductionmentioning

confidence: 99%

Mechanistic insights into intramolecular phosphate group transfer during collision induced dissociation of phosphopeptides

et al. 2019

View full text Add to dashboard Cite

We report on the rearrangement chemistry of model phosphorylated peptides during collision‐induced dissociation (CID), where intramolecular phosphate group transfers are observed from donor to acceptor residues. Such “scrambling” could result in inaccurate modification localization, potentially leading to misidentifications. Systematic studies presented herein provide mechanistic insights for the unusually high phosphate group rearrangements presented some time ago by Reid and coworkers (Proteomics 2013, 13 [6], 964‐973). It is postulated here that a basic residue like histidine can play a key role in mediating the phosphate group transfer by deprotonating the serine acceptor site. The proposed mechanism is consistent with the observation that fast collisional activation by collision‐cell CID and higher‐energy collisional dissociation (HCD) can shut down rearrangement chemistry. Additionally, the rearrangement chemistry is highly dependent on the charge state of the peptide, mirroring previous studies that less rearrangement is observed under mobile proton conditions.

show abstract

“…This constitutes a major challenge for phospho-proteomic techniques, limiting their application to clinical biopsies. However considerable methodological improvements now enable phosphoproteomic analysis of sub-mg quantities at moderate depth [21][22][23] . Limited proteolytic processing is an irreversible enzymatic modification that regulates physiological and pathological processes through inactivation or changes in function, location, and stability of proteins.…”

Section: Introductionmentioning

confidence: 99%

PDX models reflect the proteome landscape of pediatric acute lymphoblastic leukemia but divert in select pathways

Uzozie¹,

Ergin²,

Rolf³

et al. 2019

Preprint

View full text Add to dashboard Cite

Murine xenografts of pediatric leukemia are known to accurately recapitulate genomic aberrations. How this translates to the functional capacity of the proteome is unknown. Here, we studied global protein abundance, phosphorylation, and proteolytic processing in 11 pediatric B-and T-cell acute lymphoblastic leukemia patients and 19 corresponding xenografts. Protein level differences that stratified pediatric disease subtypes at diagnostic and relapse stages were largely recapitulated in xenograft models. Patient xenografts lacked multiple human leukocyte antigens, and complement proteins, and presented incomplete response mechanisms to the host immune system which is absent in the murine model. The dominant expression of MKI67 and cell cycle proteins indicated a high proliferative capacity of xenografted cells residing in the spleen. Structural genomic changes and mutations found in patients were reflected at the protein level. The post-translational modification landscape is shaped by leukemia type and host and only to a limited degree by the patient of origin. This study portrays how genomic and host factors shape protein and post-translational modification landscapes differently, and confirms murine patient-derived xenograft as competent model system while highlighting important areas of diverging biology.

show abstract

Phosphopeptide Fragmentation and Site Localization by Mass Spectrometry: An Update

Cited by 96 publications

References 164 publications

Strong anion exchange‐mediated phosphoproteomics reveals extensive human non‐canonical phosphorylation

Strong anion exchange‐mediated phosphoproteomics reveals extensive human non‐canonical phosphorylation

Mechanistic insights into intramolecular phosphate group transfer during collision induced dissociation of phosphopeptides

PDX models reflect the proteome landscape of pediatric acute lymphoblastic leukemia but divert in select pathways

Contact Info

Product

Resources

About