Improvement of Shotgun Proteomics in the Negative Mode by Carbamylation of Peptides and Ultraviolet Photodissociation Mass Spectrometry

Greer, Sylvester M.; Cannon, Joe R.; Brodbelt, Jennifer S.

doi:10.1021/ac5035314

Cited by 29 publications

(46 citation statements)

References 28 publications

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“…193 nm UVPD has been utilized in a number of proteomic applications, 103–122 including ones aimed at characterization of sites of modifications, including phosphorylation, 104–105 sulfation, 106–108 and glycosylation, 1109–110 for de novo sequencing, 111 and for extending the breadth and depth of bottom-up methods via evaluation of acidic peptides in the negative mode. 112–113 193 nm UVPD has also shown success for analysis of intact proteins and protein complexes, 116–122 both discussed in more detail in later sections.…”

Section: Photoactivationmentioning

confidence: 99%

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Ion Activation Methods for Peptides and Proteins

2015

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Section: Photoactivationmentioning

confidence: 99%

“…Activation methods geared for deprotonated peptides include electron detachment dissociation (EDD), 69–72 negative electron transfer dissociation (NETD), 76–81 electron photodetachment (EPD), 152–154 and UVPD. 105–113,155 …”

Section: Activation Of Deprotonated Peptidesmentioning

confidence: 99%

Ion Activation Methods for Peptides and Proteins

2015

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“…Another merit of UVPD is the ability to generate diagnostic product ions from peptide anions. 19–21 Although peptide analysis is generally undertaken in the positive ion mode based on greater sensitivity and number of applicable MS/MS techniques, the negative mode offers unique benefits for certain types of peptides. This is especially true for characterization of labile PTMs including phosphorylation, sulfation, and O-glycosylation, all of which exhibit superior retention using negative mode UVPD.…”

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confidence: 99%

Mapping the Phosphorylation Pattern of Drosophila melanogaster RNA Polymerase II Carboxyl-Terminal Domain Using Ultraviolet Photodissociation Mass Spectrometry

et al. 2016

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Phosphorylation of the C-terminal domain of RNA polymerase II (CTD) plays an essential role in eukaryotic transcription by recruiting transcriptional regulatory factors to the active polymerase. However, the scarcity of basic residues and repetitive nature of the CTD sequence impose a huge challenge for site-specific characterization of phosphorylation, hindering our understanding of this crucial biological process. Herein, we apply LC-UVPD-MS methods to analyze post-translational modification along native sequence CTDs. Application of our method to the Drosophila melanogaster CTD reveals the phosphorylation pattern of this model organism for the first time. The divergent nature of fly CTD allows us to derive rules defining how flanking residues affect phosphorylation choice by CTD kinases. Our data support the use of LC-UVPD-MS to decipher the CTD code and determine rules that program its function.

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“…Additionally, challenges with column longevity and hindered performance of LC pumps have been reported for high-pH reversed-phase separations for negative mode proteomics (7,24,73). We previously struggled with both precolumn and analytical column degradation due to the instability of silica in basic conditions.…”

Section: Discussionmentioning

confidence: 99%

The Negative Mode Proteome with Activated Ion Negative Electron Transfer Dissociation (AI-NETD)

Riley¹,

Rush

Rose

et al. 2015

Molecular & Cellular Proteomics

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The field of proteomics almost uniformly relies on peptide cation analysis, leading to an underrepresentation of acidic portions of proteomes, including relevant acidic posttranslational modifications. Despite the many benefits negative mode proteomics can offer, peptide anion analysis remains in its infancy due mainly to challenges with high-pH reversed-phase separations and a lack of robust fragmentation methods suitable for peptide anion characterization. Here, we report the first implementation of activated ion negative electron transfer dissociation (AI-NETD) on the chromatographic timescale, generating 7,601 unique peptide identifications from Saccharomyces cerevisiae in single-shot nLC-MS/MS analyses of tryptic peptides-a greater than 5-fold increase over previous results with NETD alone. These improvements translate to identification of 1,106 proteins, making this work the first negative mode study to identify more than 1,000 proteins in any system. We then compare the performance of AI-NETD for analysis of peptides generated by five proteases (trypsin, LysC, GluC, chymotrypsin, and AspN) for negative mode analyses, identifying as many as 5 Global protein analysis continues to enjoy substantial technological leaps forward in its ability to characterize protein expression in a variety of organisms with both speed and sensitivity (1-4). Nevertheless, the impressive advances in protein sequence technology over past decades have rigidly adhered to positive electrospray ionization for MS 1 analysis, limiting the scope of peptides and posttranslational modifications that can be analyzed. Widely utilized acidic mobile phases both permit stable and reproducible reversed-phase separations and also provide optimal conditions for ionization and detection of peptides and proteins that readily accept positive charge via protonation (i.e. basic species). Acidic peptides and proteins, however, favor deprotonation, making positive electrospray regimes ill suited for their characterization. Moreover, important classes of posttranslational modifications (PTMs), such as phosphorylation, sulfation, and glycosylation, can impart acidic properties to the peptides and proteins they modify, often producing entire classes of biomolecules that preferentially ionize as anions (5-10).Electrospray ionization operated in the negative mode can generate multiply deprotonated species (11, 12); however, canonical collisional activation methods produce MS/MS spectra riddled with neutral losses and internal fragments that are difficult, if not impossible, to interpret (13-16). AlternaFrom the ‡Department

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Improvement of Shotgun Proteomics in the Negative Mode by Carbamylation of Peptides and Ultraviolet Photodissociation Mass Spectrometry

Cited by 29 publications

References 28 publications

Ion Activation Methods for Peptides and Proteins

Ion Activation Methods for Peptides and Proteins

Mapping the Phosphorylation Pattern of Drosophila melanogaster RNA Polymerase II Carboxyl-Terminal Domain Using Ultraviolet Photodissociation Mass Spectrometry

The Negative Mode Proteome with Activated Ion Negative Electron Transfer Dissociation (AI-NETD)

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