An Automated Multidimensional Protein Identification Technology for Shotgun Proteomics

Wolters, Dirk; Washburn, Michael P.; Yates, John R.

doi:10.1021/ac010617e

Cited by 1,693 publications

(1,438 citation statements)

References 27 publications

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“…As observed in our previous studies [50][51][52][53], the pI of basic peptides may not be well defined and the basic peptides do not focus as tightly as those of acidic counterparts during the CIEP separation. In comparison with the shotgun-based multidimensional LC system [54,55], a high degree of peptide overlapping was typically observed with 40-80% of carry over peptides that were identified in the previous salt steps in the first dimension of strong-cation-exchange chromatography.…”

Section: Resultsmentioning

confidence: 99%

Application of capillary isotachophoresis‐based multidimensional separations coupled with electrospray ionization‐tandem mass spectrometry for characterization of mouse brain mitochondrial proteome

et al. 2008

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By employing a capillary ITP (CITP)/CZE-based proteomic technology, a total of 1795 distinct mouse Swiss-Prot protein entries (or 1705 nonredundant proteins) are identified from synaptic mitochondria isolated from mouse brain. The ultrahigh resolving power of CITP/CZE is evidenced by the large number of distinct peptide identifications measured from each CITP fraction together with the low peptide fraction overlapping among identified peptides. The degree of peptide overlapping among CITP fractions is even lower than that achieved using combined CIEF/nano-RP LC separations for the analysis of the same mitochondrial sample. When evaluating the protein sequence coverage by the number of distinct peptides mapping to each mitochondrial protein identification, CITP/CZE similarly achieves superior performance with 1041 proteins (58%) having 3 or more distinct peptides, 233 (13%) having 2 distinct peptides, and 521 (29%) having a single distinct peptide. The reproducibility of protein identifications is found to be around 86% by comparing proteins identified from repeated runs of the same mitochondrial sample. The analysis of the mouse mitochondrial proteome by two CITP/CZE runs results in the detection of 2095 distinct mouse Swiss-Prot protein entries (or 1992 nonredundant proteins), corresponding to 59% coverage of the updated Maestro mitochondrial reference set. The collective analysis from combined CITP/CZE and CIEF-based proteomic studies yields the identification of 2191 distinct mitochondrial protein entries (or 2082 nonredundant proteins), corresponding to 76% coverage of the MitoP2-database reference set.

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Section: Resultsmentioning

confidence: 99%

Application of capillary isotachophoresis‐based multidimensional separations coupled with electrospray ionization‐tandem mass spectrometry for characterization of mouse brain mitochondrial proteome

et al. 2008

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“…8,9 In most cases a collision-induced dissociation (CID) spectrum from a single peptide through electrospray (ESI) MS/MS is then sufficient to conclusively identify a protein. In this technique, peptides ions are sequentially selected for MS/MS from a mixture.…”

Section: Introductionmentioning

confidence: 99%

Electrochemical multi-tagging of cysteinyl peptides during microspray mass spectrometry: numerical simulation of consecutive reactions in a microchannel

Dayon

Josserand

2005

Phys. Chem. Chem. Phys.

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On-line electrogeneration of mass tags in a microspray emitter is used to quantify the number of cysteine groups in a given peptide. A finite-element simulation of the multi-step process yields the relative distribution and concentration of tags, untagged and tagged species in the microchannel before the spray event. The work focuses on the tagging of cysteine moieties in peptides or proteins by electrogenerated quinone mass probes. The main chemical parameters determining the kinetics of the labelling are assessed and discussed considering the microfluidic aspects of the process. The control of the tagging extent allows the simultaneous MS analysis of both the unmodified and modified peptide(s). The number of cysteine groups corresponds to the number of characteristic mass shifts observed from the unmodified peptide. The present theoretical work establishes the range of optimum conditions for the determination of the number of cysteine groups in peptides containing up to five cysteine groups.

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“…3,14 However, for the proteolytic digestion of proteins solubilized with FA, acid-compatible chemical reagents or enzymes, such as cyanogen bromide (CNBr) 7,18 or pepsin, 14,19,20 are commonly adopted. The fragments of CNBr cleavage are often too large for electrospray ionization (ESI)-MS analysis.…”

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confidence: 99%

Biphasic Microreactor for Efficient Membrane Protein Pretreatment with a Combination of Formic Acid Assisted Solubilization, On-Column pH Adjustment, Reduction, Alkylation, and Tryptic Digestion

et al. 2013

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Combining good dissolving ability of formic acid (FA) for membrane proteins and excellent complementary retention behavior of proteins on strong cation exchange (SCX) and strong anion exchange (SAX) materials, a biphasic microreactor was established to pretreat membrane proteins at microgram and even nanogram levels. With membrane proteins solubilized by FA, all of the proteomics sample processing procedures, including protein preconcentration, pH adjustment, reduction, and alkylation, as well as tryptic digestion, were integrated into an "SCX-SAX" biphasic capillary column. To evaluate the performance of the developed microreactor, a mixture of bovine serum albumin, myoglobin, and cytochrome c was pretreated. Compared with the results obtained by the traditional in-solution process, the peptide recovery (93% vs 83%) and analysis throughput (3.5 vs 14 h) were obviously improved. The microreactor was further applied for the pretreatment of 14 μg of membrane proteins extracted from rat cerebellums, and 416 integral membrane proteins (IMPs) (43% of total protein groups) and 103 transmembrane peptides were identified by two-dimensional nanoliquid chromatography-electrospray ionization tandem mass spectrometry (2D nano-LC-ESI-MS/MS) in triplicate analysis. With the starting sample preparation amount decreased to as low as 50 ng, 64 IMPs and 17 transmembrane peptides were identified confidently, while those obtained by the traditional insolution method were 10 and 1, respectively. All these results demonstrated that such an "SCX-SAX" based biphasic microreactor could offer a promising tool for the pretreatment of trace membrane proteins with high efficiency and throughput.

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An Automated Multidimensional Protein Identification Technology for Shotgun Proteomics

Cited by 1,693 publications

References 27 publications

Application of capillary isotachophoresis‐based multidimensional separations coupled with electrospray ionization‐tandem mass spectrometry for characterization of mouse brain mitochondrial proteome

Application of capillary isotachophoresis‐based multidimensional separations coupled with electrospray ionization‐tandem mass spectrometry for characterization of mouse brain mitochondrial proteome

Electrochemical multi-tagging of cysteinyl peptides during microspray mass spectrometry: numerical simulation of consecutive reactions in a microchannel

Biphasic Microreactor for Efficient Membrane Protein Pretreatment with a Combination of Formic Acid Assisted Solubilization, On-Column pH Adjustment, Reduction, Alkylation, and Tryptic Digestion

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