Identification of an artifact in the mass spectrometry of proteins derivatized with iodoacetamide

Lapko, Veniamin N.; Smith, David L.; Smith, Jean B.

doi:10.1002/(sici)1096-9888(200004)35:4<572::aid-jms971>3.0.co;2-2

Cited by 63 publications

(47 citation statements)

References 6 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The mass of 1852 is contributed from the peptide with methionine oxidized to methionine sulfoxide (with a mass increase of 16 Da) (5) and the asparagine from the consensus sequence modified to aspartic acid (with a mass increase of 1 Da) after PNGase F deglycosylation (28). The mass of 1791 is attributed from the same peptide as shown by the CID spectrum with a cleavage between the ␥ carbon and the sulfur of the methionine side chain, which most likely happened during the mass spectrometry direction (33)(34)(35). The successful identification of the formally glycosylated peptide from chicken avidin indicated that the capture strategy we developed here was effective and that the sodium sulfite we introduced to reduce periodate did not interfere with the capture procedure.…”

Section: Resultsmentioning

confidence: 99%

Shotgun Glycopeptide Capture Approach Coupled with Mass Spectrometry for Comprehensive Glycoproteomics

Sun

Ranish

Utleg

et al. 2007

Molecular & Cellular Proteomics

156

186

View full text Add to dashboard Cite

We present a robust and general shotgun glycoproteomics approach to comprehensively profile glycoproteins in complex biological mixtures. In this approach, glycopeptides derived from glycoproteins are enriched by selective capture onto a solid support using hydrazide chemistry followed by enzymatic release of the peptides and subsequent analysis by tandem mass spectrometry. The approach was validated using standard protein mixtures that resulted in a close to 100% capture efficiency. Our capture approach was then applied to microsomal fractions of the cisplatinresistant ovarian cancer cell line IGROV-1/CP. With a Protein Prophet probability value greater than 0.9, we identified a total of 302 proteins with an average protein identification rate of 136 ؎ 19 (n ‫؍‬ 4) in a single linear quadrupole ion trap (LTQ) mass spectrometer nano-LC-MS experiment and a selectivity of 91 ؎ 1.6% (n ‫؍‬ 4) for the N-linked glycoconsensus sequence. Our method has several advantages. 1) Digestion of proteins initially into peptides improves the solubility of large membrane proteins and exposes all of the glycosylation sites to ensure equal accessibility to capture reagents. 2) Capturing glycosylated peptides can effectively reduce sample complexity and at the same time increase the confidence of MS-based protein identifications (more potential peptide identifications per protein). 3) The utility of sodium sulfite as a quencher in our capture approach to replace the solid phase extraction step in an earlier glycoprotein chemical capture approach for removing excess sodium periodate allows the overall capture procedure to be completed in a single vessel. This improvement minimizes sample loss, increases sensitivity, and makes our protocol amenable for high throughput implementation, a feature that is essential for biomarker identification and validation of a large number of clinical samples. 4) The approach is demonstrated here on the analysis of N-linked glycopeptides; however, it can be applied equally well to

show abstract

Section: Resultsmentioning

confidence: 99%

Shotgun Glycopeptide Capture Approach Coupled with Mass Spectrometry for Comprehensive Glycoproteomics

Sun

Ranish

Utleg

et al. 2007

Molecular & Cellular Proteomics

156

186

View full text Add to dashboard Cite

show abstract

“…Sequence of phosphoribosylformylglycinamidine cycloligase (SO_2760) in Shewanella oneidensis MR-1 according to TIGR annotation (red), observed nontryptic peptide (blue) and alignment to the orthologs in other Shewanella strains (green). Lagerwerf et al 1996;Lapko et al 2000 Carbamidomethylation (abbreviated as CAM) is added to cysteine side chains by treatment with iodoacetamide, but can be attached to other sites. Most of these modifications occur in vitro.…”

Section: Chemical Modificationsmentioning

confidence: 99%

Whole proteome analysis of post-translational modifications: Applications of mass-spectrometry for proteogenomic annotation

Gupta¹,

Tanner²,

Jaitly³

et al. 2007

Genome Res.

181

225

View full text Add to dashboard Cite

While bacterial genome annotations have significantly improved in recent years, techniques for bacterial proteome annotation (including post-translational chemical modifications, signal peptides, proteolytic events, etc.) are still in their infancy. At the same time, the number of sequenced bacterial genomes is rising sharply, far outpacing our ability to validate the predicted genes, let alone annotate bacterial proteomes. In this study, we use tandem mass spectrometry (MS/MS) to annotate the proteome of Shewanella oneidensis MR-1, an important microbe for bioremediation. In particular, we provide the first comprehensive map of post-translational modifications in a bacterial genome, including a large number of chemical modifications, signal peptide cleavages, and cleavages of N-terminal methionine residues. We also detect multiple genes that were missed or assigned incorrect start positions by gene prediction programs, and suggest corrections to improve the gene annotation. This study demonstrates that complementing every genome sequencing project by an MS/MS project would significantly improve both genome and proteome annotations for a reasonable cost.

show abstract

“…With a pKa of about 8.3 for protein thiols, it is clear that the Cys thiol will be most reactive at high pH. Side reactions that may occur include alkylation of His, Lys, Met, or Tyr residues by the iodoacetamide [18]. In contrast, maleimides proceed via Michael addition where the nucleophile (thiolate) reacts with the olefin (maleimide).…”

Section: Optimization Of Labeling With Bodipy Fl-mal In the Presence mentioning

confidence: 99%

Saturation fluorescence labeling of proteins for proteomic analyses

Pretzer

Wiktorowicz

2008

Analytical Biochemistry

View full text Add to dashboard Cite

We present here an optimized and cost-effective approach to saturation fluorescence labeling of protein thiols for proteomic analysis. We investigated a number of conditions and reagent concentrations including a disulfide reducing agent (TCEP), pH, incubation time, linearity of labeling, and saturating dye: protein thiol ratio with protein standards to gauge specific and nonspecific labeling. Efficacy of labeling under these conditions was quantified using specific fluorescence estimation, defined as the ratio of fluorescence pixel intensities and Coomassie-stained pixel intensities of bands after digital imaging. Factors leading to specific vs. non-specific labeling in the presence of thiourea are also discussed.We have found that reproducible saturation of available Cys residues of the proteins used as labeling standards (human carbonic anhydrase I, enolase, α-lactalbumin) is achieved at 50-100-fold excess of the uncharged maleimide-functionalized BODIPY™ dyes over Cys. We confirm our previous findings and those of others that the maleimide dyes are not impacted by the presence of 2M thiourea. Moreover, we establish that 2 mM TCEP used as reductant is optimal. We also establish further that labeling is optimal at pH 7.5 and complete after 30 min. Low non-specific labeling was gauged by the inclusion of non-Cys containing proteins (horse myoglobin, bovine carbonic anhydrase) to the labeling mixture. We also show that the dye exhibits little to no effect on the two dimensional mobilities of labeled proteins derived from cells.

show abstract

Identification of an artifact in the mass spectrometry of proteins derivatized with iodoacetamide

Cited by 63 publications

References 6 publications

Shotgun Glycopeptide Capture Approach Coupled with Mass Spectrometry for Comprehensive Glycoproteomics

Shotgun Glycopeptide Capture Approach Coupled with Mass Spectrometry for Comprehensive Glycoproteomics

Whole proteome analysis of post-translational modifications: Applications of mass-spectrometry for proteogenomic annotation

Saturation fluorescence labeling of proteins for proteomic analyses

Contact Info

Product

Resources

About