Electron Transfer/Higher Energy Collisional Dissociation of Doubly Charged Peptide Ions: Identification of Labile Protein Phosphorylations

Penkert, Martin; Hauser, Anett; Harmel, Robert K.; Fiedler, Dorothea; Hackenberger, Christian P. R.; Krause, E.

doi:10.1007/s13361-019-02240-4

Cited by 24 publications

(20 citation statements)

References 46 publications

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“…However, additional efforts by the proteomics research community are required to define the best resolution and fragmentation methods for non-conventional phosphosites and develop new technology compatible with identification of the full range of phosphorylation events. In this regard, the electron transfer/higher energy collisional dissociation (EThcD) has recently been reported to fragment doubly-charged phosphopeptides without loss of the labile phosphates on Arg, His, Cys, and Lys as well as Ser residues using synthetic peptides 27 . In addition, the development of nETD (negative ion mode) fragmentation and MS analysis, where the polarity is reversed, should be highly compatible with a non-acidic phosphoproteomic analysis 28 .…”

Section: Discussionmentioning

confidence: 99%

A non-acidic method using hydroxyapatite and phosphohistidine monoclonal antibodies allows enrichment of phosphopeptides containing non-conventional phosphorylations for mass spectrometry analysis

Adam

Fuhs

Meisenhelder

et al. 2019

Preprint

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Four types of phosphate-protein linkage generate nine different phosphoresidues in living organisms. Histidine phosphorylation is a long-time established but largely unexplored post-translational modification, mainly because of the acid-lability of the phosphoramidate bonds. This lability means that standard phosphoproteomic methods used for conventional phosphate esters (phospho-Ser/Thr/Tyr) must be modified to analyze proteins containing the phosphoramidate-amino acids -phospho-His/Arg/Lys. We show that a non-acidic method allows enrichment of non-conventional phosphoresiduecontaining peptides from tryptic digests of human cell lines, using hydroxyapatite binding and/or immobilized 1-pHis and 3-pHis monoclonal antibodies for enrichment. 425 unique non-conventional phosphorylation sites (i.e. pHis, pLys and pArg) were detected with a high probability of localization by LC-MS/MS analysis and identified using a customized MaxQuant configuration, contributing to a new era of study in post-translational modification and cell signaling in humans. This is the first fully non-acidic method for phosphopeptide enrichment which uses immunoaffinity purification and remains compatible with mass spectrometry analysis for a wider coverage of potential protein phosphorylation events. Introduction:Phosphoproteomics has contributed to tremendous progress in the field of life science and revealed the extensive use of phosphorylation as one of the most important post-translational modifications in eukaryotic organisms. The identification and characterization of phosphorylation sites has been revolutionized by mass spectrometry (MS) with faster and more sensitive instruments. Progress in phosphosite-mapping with tandem mass spectrometry (MS/MS) has allowed improved localization and phosphorylation assignment to a single residue with high confidence. The development of bioinformatics tools for large-scale phosphoproteome data analysis was an equally important step and permitted the integration of different parameters searching for complex combinatorial possibilities 1,2 .Up until now, only one class of phosphate-bond containing phosphoamino acid, corresponding to the conventional serine, threonine and tyrosine phosphorylation (phosphate-ester or O-phosphate) has been systematically considered for phosphorylation site searching. But nine out of the 20 amino acids have the

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Section: Discussionmentioning

confidence: 99%

A non-acidic method using hydroxyapatite and phosphohistidine monoclonal antibodies allows enrichment of phosphopeptides containing non-conventional phosphorylations for mass spectrometry analysis

Adam

Fuhs

Meisenhelder

et al. 2019

Preprint

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“…Potentially, the observation of the GMP-modified ser-2 on recombinant SARS-CoV-2 nsp7 in the mass spectrometer could be the manifestation of a lys-3 GMP-modification via a phosphoramide bond that was transferred to a more energetically favorable position on the neighboring residue during the sample handling. It should be noted that non-canonical phosphorylation on basic amino acids, which uses identical phosphoramidate chemistry (i.e., the P-N bond versus P-O in S, T and Y amino acids) for the GMP linkage, is beginning to emerge as an important component of signaling systems in nature 16,23 . These phosphoramidate bonds are more labile under the conditions commonly used in analytical instrumentation such as HPLC and mass spectrometry and are therefore understudied.…”

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confidence: 99%

Mass spectrometric based detection of protein nucleotidylation in the RNA polymerase of SARS-CoV-2

Conti

Kirchdoerfer

Sussman

2020

Preprint

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SummaryCoronaviruses, like SARS-CoV-2, encode a nucleotidyl transferase in the N-terminal NiRAN domain of the non-structural protein (nsp) 12 protein within the RNA dependent RNA polymerase (RdRP) 1-3. Though the substrate targets of the viral nucleotidyl transferase are unknown, NiRAN active sites are highly conserved and essential for viral replication 3. We show, for the first time, the detection and sequence location of GMP-modified amino acids in nidovirus RdRP-associated proteins using heavy isotope-assisted MS and MS/MS peptide sequencing. We identified lys-143 in the equine arteritis virus (EAV) protein, nsp7, as a primary site of nucleotidylation in vitro that uses a phosphoramide bond to covalently attach with GMP. In SARS-CoV-2 replicase proteins, we demonstrate a unique O-linked GMP attachment on nsp7 ser-1, whose formation required the presence of nsp12. It is clear that additional nucleotidylation sites remain undiscovered, which includes the possibility that nsp12 itself may form a transient GMP adduct in the NiRAN active site that has eluted detection in these initial studies due to instability of the covalent attachment. Our results demonstrate new strategies for detecting GMP-peptide linkages that can be adapted for higher throughput screening using mass spectrometric technologies. These data are expected to be important for a rapid and timely characterization of a new enzymatic activity in SARS-CoV-2 that may be an attractive drug target aimed at limiting viral replication in infected patients.

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“…For pHis and pArg, extensive neutral loss of H 3 PO 4 or HPO 3, as well as phosphoryl transfer to other residues, were observed during collision‐induced dissociation (CID) or higher‐energy collisional dissociation (HCD) processes, leading to difficult phosphosite localization [26,43–45] . Recently, electron‐transfer/higher‐energy collisional dissociation (EThCD) was shown to preserve pLys, pCys, pArg, pHis, ppSer, and ppThr sites during the fragmentation to improve the phosphosite localization significantly [46,47] . EThCD generally suffers from longer duty cycle and lower sensitivity than other fragmentation methods, but CID‐triggered EThCD, successfully demonstrated for the analysis of ppSer and ppThr, [48] may overcome those limitations.…”

Section: Preservation Enrichment and Analysis Of The Phosphosites Imentioning

confidence: 99%

“…[26,[43][44][45] Recently, electron-transfer/higher-energy collisional dissociation (EThCD) was shown to preserve pLys, pCys, pArg, pHis, ppSer, and ppThr sites during the fragmentation to improve the phosphosite localization significantly. [46,47] EThCD generally suffers from longer duty cycle and lower sensitivity than other fragmentation methods, but CID-triggered EThCD, successfully demonstrated for the analysis of ppSer and ppThr, [48] may overcome those limitations. Given the remarkable technological advances described above, we can expect that an optimized standard phosphoproteomics workflow for the "crypto-phosphoproteome" will emerge soon.…”

Section: Preservation Enrichment and Analysis Of The Phosphosites Imentioning

confidence: 99%

Quest for the Crypto‐phosphoproteome

2020

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Protein phosphorylation is one of the most studied posttranslational modifications (PTMs). Despite the remarkable advances in phosphoproteomics, a chemically less-stable subset of the phosphosites, which we call the crypto-phosphopro-teome, has remained underexplored due to technological challenges. In this Viewpoint, we briefly summarize the current understanding of these elusive protein phosphorylations and identify the missing pieces for future studies.

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Electron Transfer/Higher Energy Collisional Dissociation of Doubly Charged Peptide Ions: Identification of Labile Protein Phosphorylations

Cited by 24 publications

References 46 publications

A non-acidic method using hydroxyapatite and phosphohistidine monoclonal antibodies allows enrichment of phosphopeptides containing non-conventional phosphorylations for mass spectrometry analysis

A non-acidic method using hydroxyapatite and phosphohistidine monoclonal antibodies allows enrichment of phosphopeptides containing non-conventional phosphorylations for mass spectrometry analysis

Mass spectrometric based detection of protein nucleotidylation in the RNA polymerase of SARS-CoV-2

Quest for the Crypto‐phosphoproteome

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