Methods for the Detection of Paxillin Post-translational Modifications and Interacting Proteins by Mass Spectrometry

Schroeder, Melanie; Webb, Donna J.; Shabanowitz, Jeffrey; Horwitz, and Alan F.; Hunt, Donald F.

doi:10.1021/pr0502020

Cited by 66 publications

(61 citation statements)

References 63 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Electron transfer dissociation (ETD) was developed in an attempt to find an ECD-like dissociation method, which could be used on widely accessible instruments, such as quadrupole linear ITs [118,119]. Syka and co-workers found that anions with sufficiently low electron affinities (e.g., anthracene or azobenzene) could function as one-electron donors, and induce fragmentation by the same nonergodic process as ECD [119].…”

Section: Electron Transfer Dissociationmentioning

confidence: 99%

Analytical strategies for phosphoproteomics

2009

View full text Add to dashboard Cite

Protein phosphorylation is a key regulator of cellular signaling pathways. It is involved in most cellular events in which the complex interplay between protein kinases and protein phosphatases strictly controls biological processes such as proliferation, differentiation, and apoptosis. Defective or altered signaling pathways often result in abnormalities leading to various diseases, emphasizing the importance of understanding protein phosphorylation. Phosphorylation is a transient modification, and phosphoproteins are often very low abundant. Consequently, phosphoproteome analysis requires highly sensitive and specific strategies. Today, most phosphoproteomic studies are conducted by mass spectrometric strategies in combination with phosphospecific enrichment methods. This review presents an overview of different analytical strategies for the characterization of phosphoproteins. Emphasis will be on the affinity methods utilized specifically for phosphoprotein and phosphopeptide enrichment prior to MS analysis, and on recent applications of these methods in cell biological applications.

show abstract

Section: Electron Transfer Dissociationmentioning

confidence: 99%

Analytical strategies for phosphoproteomics

2009

View full text Add to dashboard Cite

show abstract

“…The use of optimized neutral loss settings where the first two losses of phosphoric acid would be ignored would favor triphosphorylated peptides and would be expected to shift the distribution of multiply phosphorylated peptides. Replacing CID with ECD/ETD for fragmentation would evade this problem because this method primarily results in peptide backbone fragmentation without concomitant loss of the phosphate groups (22,23).…”

Section: A Modified Pdmsmentioning

confidence: 99%

“…Presently manual inspection showed that only a small fraction of the phosphorylated peptide ions detected by LC-MSMS is identified using the Mascot database search software (Ͻ30%). To increase the number of identified phosphopeptides, the SIMAC strategy could be combined with multistage activation (28) or ETD (23), which would provide a better coverage of the peptide sequence.…”

Section: A Modified Pdmsmentioning

confidence: 99%

“…Unfortunately this strategy favors the analysis of monophosphorylated peptides because multiply phosphorylated peptides will subsequently lose more phosphoric acid. Electron capture/transfer dissociation (ECD/ETD) is an emerging alternate technology that can be applied to multiply phosphorylated peptides because this method primarily results in peptide backbone fragmentation without concomitant loss of the phosphate groups (22,23). However, ECD/ETD is not yet readily available in most research laboratories.…”

mentioning

confidence: 99%

See 1 more Smart Citation

SIMAC (Sequential Elution from IMAC), a Phosphoproteomics Strategy for the Rapid Separation of Monophosphorylated from Multiply Phosphorylated Peptides

Thingholm

Jensen

Robinson

et al. 2008

Molecular & Cellular Proteomics

399

415

View full text Add to dashboard Cite

The complete analysis of phosphoproteomes has been hampered by the lack of methods for efficient purification, detection, and characterization of phosphorylated peptides from complex biological samples. Despite several strategies for affinity enrichment of phosphorylated peptides prior to mass spectrometric analysis, such as immobilized metal affinity chromatography or titanium dioxide, the coverage of the phosphoproteome of a given sample is limited. Here we report a simple and rapid strategy, SIMAC (sequential elution from IMAC), for sequential separation of monophosphorylated peptides and multiply phosphorylated peptides from highly complex biological samples. This allows individual analysis of the two pools of phosphorylated peptides using mass spectrometric parameters differentially optimized for their unique properties. We compared the phosphoproteome identified from 120 g of human mesenchymal stem cells using SIMAC and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SI-

show abstract

“…The ETD spectrum from a 55 amino acid tryptic peptide provided the necessary fragment ions to assign Ser 74 of paxillin as being modified by 203 amu, corresponding to the mass of GlcNAc [34]. The CAD spectrum of this peptide produced highly charged fragment ions with a loss of 203 amu.…”

Section: O-and N-linked Glycosylationmentioning

confidence: 99%

The utility of ETD mass spectrometry in proteomic analysis

Mikesh

Ueberheide

Chen

et al. 2006

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

Self Cite

497

490

View full text Add to dashboard Cite

Mass spectrometry has played an integral role in the identification of proteins and their posttranslational modifications (PTM). However, analysis of some PTMs, such as phosphorylation, sulfonation, and glycosylation, is difficult with collision-activated dissociation (CAD) since the modification is labile and preferentially lost over peptide backbone fragmentation, resulting in little to no peptide sequence information. The presence of multiple basic residues also makes peptides exceptionally difficult to sequence by conventional CAD mass spectrometry. Here we review the utility of electron transfer dissociation (ETD) mass spectrometry for sequence analysis of posttranslationally modified and/or highly basic peptides. Phosphorylated, sulfonated, glycosylated, nitrosylated, disulfide bonded, methylated, acetylated, and highly basic peptides have been analyzed by CAD and ETD mass spectrometry. CAD fragmentation typically produced spectra showing limited peptide backbone fragmentation. However, when these peptides were fragmented using ETD, peptide backbone fragmentation produced a complete or almost complete series of ions and thus extensive peptide sequence information. In addition, labile PTMs remained intact. These examples illustrate the utility of ETD as an advantageous tool in proteomic research by readily identifying peptides resistant to analysis by CAD. A further benefit is the ability to analyze larger, non-tryptic peptides, allowing for the detection of multiple PTMs within the context of one another.

show abstract

Methods for the Detection of Paxillin Post-translational Modifications and Interacting Proteins by Mass Spectrometry

Cited by 66 publications

References 63 publications

Analytical strategies for phosphoproteomics

Analytical strategies for phosphoproteomics

SIMAC (Sequential Elution from IMAC), a Phosphoproteomics Strategy for the Rapid Separation of Monophosphorylated from Multiply Phosphorylated Peptides

The utility of ETD mass spectrometry in proteomic analysis

Contact Info

Product

Resources

About