A hybrid LC–Gel-MS method for proteomics research and its application to protease functional pathway mapping

Wu, Si; Tang, Xiao Ting; Siems, William F.; Bruce, James E.

doi:10.1016/j.jchromb.2005.05.043

Cited by 11 publications

(9 citation statements)

References 42 publications

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“…The purpose of using SDS-PAGE was just to facilitate protease digestion. It is believed that proteins in such a denaturing gel matrix will be in an open structure, which increases the tryptic digestion efficiency. , After trypsinization, peptides were extracted from the gel and subjected to nanoscale LC−ESI−MS/MS. The base-peak chromatogram for reversed-phase chromatography monitored by the mass spectrometer represents the intensity of all peptide ions in the sample in a single scan (Figure ).…”

Section: Resultsmentioning

confidence: 99%

Identification of Protein Components in in vivo Human Acquired Enamel Pellicle Using LC−ESI−MS/MS

et al. 2007

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The acquired enamel pellicle is a thin protein film forming upon exposure of tooth enamel surfaces to saliva. The structural analysis of this integument relies on efficient pellicle harvesting and protein identification procedures. Material from three individual subjects and two pooled samples yielded the identification by LC-ESI-MS/MS of 130 pellicle proteins of which 89 were found in three or more experiments. A high intersubject consistency in pellicle composition was observed.

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Section: Resultsmentioning

confidence: 99%

Identification of Protein Components in in vivo Human Acquired Enamel Pellicle Using LC−ESI−MS/MS

et al. 2007

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“…The strategy takes advantage of selective blocking of lysine side-chain amines and unique amino-specific stable isotopic labeling reagents known as iTRAQ reagents. It is complementary to the combined fractional diagonal chromatography (COFRADIC) 26 or other similar approaches, 27 which utilizes peptides isotopically labeled by digestive protease-mediated incorporation of O into the C-terminus. 28 COFRADIC requires sequential LC separations prior to MS, whereas the iTRAQ procedure obviates the need for separations, thus simplifying proteome analysis.…”

Section: Discussionmentioning

confidence: 99%

Identification of Proteolytic Cleavage Sites by Quantitative Proteomics

Enoksson

Ivancic

et al. 2007

J. Proteome Res.

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The identification of natural substrates and their cleavage sites is pivotal to defining proteolytic pathways. Here we report a novel strategy for the identification of the signature of proteolytic cleavage events based on quantitative proteomics. Lysine residues in proteins are blocked by guanidination so that free N-terminals can be labeled with amine-specific iTRAQ reagents. The quantitative nature of iTRAQ reagents allows us to distinguish N-terminals newly formed by proteolytic treatment (neoepitopes) from original N-terminals in proteins. Proteins are digested with trypsin and analyzed using MALDI-TOF/TOF mass spectrometry. Peptides labeled with iTRAQ reagents are distinguished from other peptides by exhibiting intense signature ions in tandem mass spectrometry analysis. A corresponding data acquisition strategy was developed to specifically analyze iTRAQ tagged N-terminal peptides. To validate the procedure, we examined a set of recombinant Escherichia coli proteins that have predicted caspase-3 cleavage motifs. The protein mixture was treated with active or inactive caspase-3 and subsequently labeled with two different iTRAQ reagents. Mass spectrometric analysis located 10 cleavage sites, all corresponding to caspase-3 consensus. Spiking caspase-cleaved substrate into a human cell lysate demonstrated the high sensitivity of the procedure. Moreover, we were able to identify proteolytic cleavage products associated with the induction of cell-free apoptosis. Together, these data reveal a novel application for iTRAQ technology for the detection of proteolytic substrates.

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“…Other two-dimensional approaches to protein fractionation have also been proposed combining, for example, continuous-elution gel electrophoresis and reversed phase HPLC [33] or anion exchange chromatography and 1D SDS-PAGE electrophoresis. [20,28] Another 2D chromatographic strategy termed multidimensional protein identification technology (MudPIT) has been extensively applied to proteomics analyses, although at a peptide level. It consists in, first, digesting the whole protein sample and then separating the peptide mixture by one or two dimensions of chromatography prior to LC-MS analysis.…”

Section: Introductionmentioning

confidence: 99%

“…Diverse chromatography techniques have been extensively described in the literature as methods for protein/peptide separation. Cation exchange, anion exchange, reversed-phase, − biphasic ion-exchange, chromatofocusing, − or size-exclusion are all used for protein separation. As with electrophoresis, complex samples often need to be pre-fractionated 30 or separated by two dimensions of chromatography (2D HPLC), to obtain appropriately simplified samples for MS analysis. ,, These 2D HPLC technologies typically combine two of the chromatography techniques previously listed.…”

Section: Introductionmentioning

confidence: 99%

Protein Separation and Characterization by np-RP−HPLC Followed by Intact MALDI-TOF Mass Spectrometry and Peptide Mass Mapping Analyses

et al. 2006

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Due to their complexity, the separation of intact proteins from complex mixtures is an important step to comparative proteomics and the identification and characterization of the proteins by mass spectrometry (MS). In the study reported, we evaluated the use of non-porous-reversed-phase (np-RP) HPLC for intact protein separation prior to MS analyses. The separation system was characterized and compared to 1D-SDS-PAGE electrophoresis in terms of resolution and sensitivity. We demonstrate that np-RP HPLC protein separation is highly reproducible and provides intact protein fractions which can be directly analyzed by MALDI-TOF MS for intact molecular weight determination. An in-well digestion protocol was developed, allowing for rapid protein identification by peptide mass fingerprinting (PMF) and resulted in comparable or improved peptide recovery compared with in-gel digestion. The np-RP sensitivity of detection by UV absorbance at 214 nm for intact proteins was at the low ng level and the sensitivity of peptide analysis by MALDI-TOF MS was in the 10-50 pg level. A membrane protein fraction was characterized to demonstrate application of this methodology. Among the identified proteins, multiple forms of vimentin were observed. Overall we demonstrate that np-RP HPLC followed by MALDI-TOF MS allows for rapid, sensitive and reproducible protein fractionation and very specific protein characterization by integration of PMF analysis with MS intact molecular weight information. Keywordsnon-porous reversed phase HPLC; protein liquid chromatography; peptide mass fingerprint; intact protein mass spectrometry

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A hybrid LC–Gel-MS method for proteomics research and its application to protease functional pathway mapping

Cited by 11 publications

References 42 publications

Identification of Protein Components in in vivo Human Acquired Enamel Pellicle Using LC−ESI−MS/MS

Identification of Protein Components in in vivo Human Acquired Enamel Pellicle Using LC−ESI−MS/MS

Identification of Proteolytic Cleavage Sites by Quantitative Proteomics

Protein Separation and Characterization by np-RP−HPLC Followed by Intact MALDI-TOF Mass Spectrometry and Peptide Mass Mapping Analyses

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