193 nm Ultraviolet Photodissociation Mass Spectrometry for Phosphopeptide Characterization in the Positive and Negative Ion Modes

Robinson, Michelle; Taliaferro, Juliana M.; Dalby, Kevin N.; Brodbelt, Jennifer S.

doi:10.1021/acs.jproteome.6b00289

Cited by 43 publications

(65 citation statements)

References 62 publications

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“…We calculated a percent of total phosphoryl group retention, analogous to the calculations performed for UVPD spectra of phosphopeptides, 23 to understand what fraction of product ions retain their phosphate moiety under different conditions (Figure 4c). Percent phosphoryl group retention represents the ratio of product ions ( i.e.…”

Section: Resultsmentioning

confidence: 99%

“…23,88,98–100 Negative electron transfer dissociation (NETD) is a valuable method for sequencing peptide anions in shotgun proteomic experiments, but it suffers from analogous charge-density limitations as positive ETD. 101–103 This is especially noteworthy because doubly deprotonated precursors ( i.e ., low charge density precursors) are the main ion population in negative electrospray, even more so than in positive mode.…”

Section: Resultsmentioning

confidence: 99%

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Phosphoproteomics with Activated Ion Electron Transfer Dissociation

Riley¹,

Hebert²,

Dürnberger

et al. 2017

Anal. Chem.

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The ability to localize phosphosites to specific amino acid residues is crucial to translating phosphoproteomic data into biological meaningful contexts. In a companion manuscript we described a new implementation of activated ion electron transfer dissociation (AI-ETD) on a quadrupole-Orbitrap-linear ion trap hybrid MS system (Orbitrap Fusion Lumos), which greatly improved peptide fragmentation and identification over ETD and other supplemental activation methods. Here we present the performance of AI-ETD for identifying and localizing sites of phosphorylation in both phosphopeptides and intact phosphoproteins. Using 90-minute analyses we show that AI-ETD can identify 24,503 localized phosphopeptide spectral matches enriched from mouse brain lysates, which more than triples identifications from standard ETD experiments and outperforms ETcaD and EThcD as well. AI-ETD achieves these gains through improved quality of fragmentation and MS/MS success rates for all precursor charge states, especially for doubly protonated species. We also evaluate the degree to which phosphate neutral loss occurs from phosphopeptide product ions due to the infrared photo-activation of AI-ETD and show that modifying phosphoRS (a phosphosite localization algorithm) to include phosphate neutral losses can significantly improve localization in AI-ETD spectra. Finally, we demonstrate the utility of AI-ETD in localizing phosphosites in α-casein, a ~23.5 kilodalton phosphoprotein that showed eight of nine known phosphorylation sites occupied upon intact mass analysis. AI-ETD provided the greatest sequence coverage for all five charge states investigated and was the only fragmentation method to localize all eight phosphosites for each precursor. Overall, this work highlights the analytical value AI-ETD can bring to both bottom-up and top-down phosphoproteomics.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Phosphoproteomics with Activated Ion Electron Transfer Dissociation

Riley¹,

Hebert²,

Dürnberger

et al. 2017

Anal. Chem.

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“…32, 33 UVPD exhibits impressive performance for both denatured proteins and native-like proteins, the latter typically generated in low charge states which have proven more challenging to characterize by collision- and electron-based methods. 32–36 To date, UVPD has only had limited usage for high-throughput top-down analysis of complex protein mixtures, such as cell lysates. 10 Previously, Cannon et al .…”

Section: Introductionmentioning

confidence: 99%

High-Throughput Analysis of Intact Human Proteins Using UVPD and HCD on an Orbitrap Mass Spectrometer

et al. 2017

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The analysis of intact proteins (top-down strategy) by mass spectrometry has great potential to elucidate proteoform variation, including patterns of post-translational modifications (PTMs), which may not be discernable by analysis of peptides alone (bottom-up approach). To maximize sequence coverage and localization of PTMs, various fragmentation modes have been developed to produce fragment ions from deep within intact proteins. Ultraviolet photodissociation (UVPD) has recently been shown to produce high sequence coverage and PTM retention on a variety of proteins, with increasing evidence of efficacy on a chromatographic time scale. However, utilization of UVPD for high-throughput top-down analysis to date has been limited by bioinformatics. Here, we detected 153 proteins and 489 proteoforms using UVPD and 271 proteins and 982 proteoforms using higher-energy collisional dissociation (HCD) in a comparative analysis of HeLa whole-cell lysate by qualitative top-down proteomics. Of the total detected proteoforms, 286 overlapped between the UVPD and HCD datasets, with 68% of proteoforms having C-scores greater than 40 for UVPD and 63% for HCD. The average sequence coverage (28% ± 20% for UVPD versus 17% ± 8% for HCD, p < 0.0001) found to be higher for UVPD than HCD, and with a trend toward improvement in q-value for the UVPD dataset. This study demonstrates the complementarity of UVPD and HCD for more extensive protein profiling and proteoform characterization.

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“…Despite the fact that ETD fragments the phosphopeptides without generating a neutral loss peak in the spectra, the poor dissociation of low charge density peptides hampers phosphopeptide identification. Recently, hybrid electron-transfer and high-energy collision dissociation (EThcD) fragmentation 85 and ultraviolet photodissociation (UVPD) 86, 87 have been utilized for large-scale phosphopeptide identification. Although these technologies did not show significant identification improvement, they provided complementary coverage over ETD in terms of sequence information and site localization.…”

Section: Tandem Ms Technologiesmentioning

confidence: 99%

Recent advances in phosphoproteomics and application to neurological diseases

Arrington

Hsu

Elder

et al. 2017

Analyst

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Phosphorylation has an incredible impact on the biological behavior of proteins, altering everything from intrinsic activity to cellular localization and complex formation. It is no surprise then that this post-translational modification has been the subject of intense study and that, with the advent of faster, more accurate instrumentation, the number of large-scale mass spectrometry-based phosphoproteomic studies has swelled over the past decade. Recent developments in sample preparation, phosphorylation enrichment, quantification, and data analysis strategies permit both targeted and ultra-deep phosphoproteome profiling, but challenges remain in pinpointing biologically relevant phosphorylation events. We describe here technological advances that have facilitated phosphoproteomic analysis of cells, tissues, and biofluids and note applications to neuropathologies in which the phosphorylation machinery may be dysregulated, much as it is in cancer.

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193 nm Ultraviolet Photodissociation Mass Spectrometry for Phosphopeptide Characterization in the Positive and Negative Ion Modes

Cited by 43 publications

References 62 publications

Phosphoproteomics with Activated Ion Electron Transfer Dissociation

Phosphoproteomics with Activated Ion Electron Transfer Dissociation

High-Throughput Analysis of Intact Human Proteins Using UVPD and HCD on an Orbitrap Mass Spectrometer

Recent advances in phosphoproteomics and application to neurological diseases

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