Identification of Disulfide Bonds in Protein Proteolytic Degradation Products Using <i>de novo</i>-Protein Unique Sequence Tags Approach

Shen, Yufeng; Tolić, Nikola; Smith, Richard D.

doi:10.1021/pr1002559

Cited by 6 publications

(5 citation statements)

References 27 publications

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“…To generate the AMT tag database, multiple LC‐MS/MS analyses were performed on LTQ‐Orbitrap Velos using several types of fragmentation (CID, HCD, and ETD). Proteoforms were identified using a combination of several search alghoritms, including ProSight PC , UStags approach , and MSAlign+ . Initial attempts to identify proteins using ProSight PC's “absolute mass” mode to search against the annotated human top‐down database were unsuccessful likely due to the presence of novel PTMs and/or N‐terminal signaling peptide removal .…”

Section: Resultsmentioning

confidence: 99%

Quantitative analysis of human salivary gland‐derived intact proteome using top‐down mass spectrometry

Brown

Tolić

et al. 2014

Proteomics

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There are several notable challenges inherent for fully characterizing the entirety of the human saliva proteome using bottom-up approaches, including polymorphic isoforms, PTMs, unique splice variants, deletions, and truncations. To address these challenges, we have developed a top-down based LC-MS/MS approach, which cataloged 20 major human salivary proteins with a total of 83 proteoforms, containing a broad range of PTMs. Among these proteins, several previously reported disease biomarker proteins were identified at the intact protein level, such as beta-2 microglobulin. In addition, intact glycosylated proteoforms of several saliva proteins were also characterized, including intact N-glycosylated protein prolactin inducible protein and O-glycosylated acidic protein rich protein. These characterized proteoforms constitute an intact saliva proteoform database, which was used for quantitative comparison of intact salivary proteoforms among six healthy individuals. Human parotid and submandibular/sublingual gland secretion samples (2 μg of protein each) from six healthy individuals were compared using RPLC coupled with the 12T FT-ICR mass spectrometer. Significantly different proteoform profiles were resolved with high reproducibility between parotid secretion and submandibular/sublingual glands. The results from this study provide further insight into the potential mechanisms of PTM pathways in oral glandular secretion, expanding our knowledge of this complex yet easily accessible fluid. Intact protein LC-MS approach presented herein can potentially be applied for rapid and accurate identification of biomarkers from only a few microliters of human glandular saliva.

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Section: Resultsmentioning

confidence: 99%

Quantitative analysis of human salivary gland‐derived intact proteome using top‐down mass spectrometry

Brown

Tolić

et al. 2014

Proteomics

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“…The identification false rate was estimated using a decoy dataset that was constructed by reversing each protein sequence in the human sequence database; no random hits were obtained using the UStags as the identification criteria. The identification of disulfide bond-containing peptides was completed with the method reported elsewhere [40] . The protein annotations and information from EBI ( http://www.ebi.ac.uk/ ) and Swiss-Prot ( http://ca.expasy.org ) were used to describe the proteins (substrates) identified in this work.…”

Section: Methodsmentioning

confidence: 99%

Blood Peptidome-Degradome Profile of Breast Cancer

et al. 2010

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BackgroundCancer invasion and metastasis are closely associated with activities within the degradome; however, little is known about whether these activities can be detected in the blood of cancer patients.Methodology and Principal FindingsThe peptidome-degradome profiles of pooled blood plasma sampled from 15 breast cancer patients (BCP) and age, race, and menopausal status matched control healthy persons (HP) were globally characterized using advanced comprehensive separations combined with tandem Fourier transform mass spectrometry and new data analysis approaches that facilitated top-down peptidomic analysis. The BCP pool displayed 71 degradome protein substrates that encompassed 839 distinct peptidome peptides. In contrast, the HP 50 degradome substrates found encompassed 425 peptides. We find that the ratios of the peptidome peptide relative abundances can vary as much as >4000 fold between BCP and HP. The experimental results also show differential degradation of substrates in the BCP sample in their functional domains, including the proteolytic and inhibitory sites of the plasmin-antiplasmin and thrombin-antithrombin systems, the main chains of the extracellular matrix protection proteins, the excessive degradation of innate immune system key convertases and membrane attack complex components, as well as several other cancer suppressor proteins.ConclusionsDegradomics-peptidomics profiling of blood plasma is highly sensitive to changes not evidenced by conventional bottom-up proteomics and potentially provides unique signatures of possible diagnostic utility.

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“…In this work, we investigated CID, ETD, and HCD performance as part of our efforts to develop approaches for more effectively studying aberrant protein degradomic activity associated with diseases, for example, breast cancer. − Considering potential preferences/biases of peptide identification methods, we investigated these fragmentation methods using a large range of peptide lengths and termini and applied different database search and peptide validation methods with multiple parameters. Importantly, the collective results from this joint comparison of fragmentation performance and peptide identification methods are useful for determining which combinations can improve identification rates for peptidomic-degradomic analyses and proteomics applications in general.…”

Section: Introductionmentioning

confidence: 99%

Effectiveness of CID, HCD, and ETD with FT MS/MS for Degradomic-Peptidomic Analysis: Comparison of Peptide Identification Methods

et al. 2011

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We report on the effectiveness of CID, HCD, and ETD for LC-FT MS/MS analysis of peptides using a tandem linear ion trap-Orbitrap mass spectrometer. A range of software tools and analysis parameters were employed to explore the use of CID, HCD, and ETD to identify peptides isolated from human blood plasma without the use of specific “enzyme rules”. In the evaluation of an FDR-controlled SEQUEST scoring method, the use of accurate masses for fragments increased the numbers of identified peptides (by ~50%) compared to the use of conventional low accuracy fragment mass information, and CID provided the largest contribution to the identified peptide datasets compared to HCD and ETD. The FDR-controlled Mascot scoring method provided significantly fewer peptide identifications than with SEQUEST (by 1.3–2.3 fold) at the same confidence levels, and CID, HCD, and ETD provided similar contributions to identified peptides. Evaluation of de novo sequencing and the UStags method for more intense fragment ions revealed that HCD afforded more sequence consecutive residues (e.g., ≥7 amino acids) than either CID or ETD. Both the FDR-controlled SEQUEST and Mascot scoring methods provided peptide datasets that were affected by the decoy database and mass tolerances applied (e.g., the identical peptides between the datasets could be limited to ~70%), while the UStags method provided the most consistent peptide datasets (>90% overlap) with extremely low (near zero) numbers of false positive identifications. The m/z ranges in which CID, HCD, and ETD contributed the largest number of peptide identifications were substantially overlapping. This work suggests that the three peptide ion fragmentation methods are complementary, and that maximizing the number of peptide identifications benefits significantly from a careful match with the informatics tools and methods applied. These results also suggest that the decoy strategy may inaccurately estimate identification FDRs.

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Identification of Disulfide Bonds in Protein Proteolytic Degradation Products Using de novo-Protein Unique Sequence Tags Approach

Cited by 6 publications

References 27 publications

Quantitative analysis of human salivary gland‐derived intact proteome using top‐down mass spectrometry

Quantitative analysis of human salivary gland‐derived intact proteome using top‐down mass spectrometry

Blood Peptidome-Degradome Profile of Breast Cancer

Effectiveness of CID, HCD, and ETD with FT MS/MS for Degradomic-Peptidomic Analysis: Comparison of Peptide Identification Methods

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