Protein and Peptide Sequence Analysis by Tandem Mass Spectrometry in Combination with Either Capillary Electrophoresis or Micro-Capillary HPLC

Hunt, Donald F.; Shabanowitz, Jeffrey; Moseley, M. Arthur; McCormack, Ashley L.; Michel, Hanspeter; Martino, Paul A.; Tomer, Kenneth B.; Jorgenson, James W.

doi:10.1007/978-3-0348-5678-2_26

Cited by 14 publications

(15 citation statements)

References 11 publications

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“…Contrary to the HEPES buffer used before, 13,14 the phosphate buffer used here can readily be washed out from the PC with excess water. The remaining SPA and its hydrolysis products were removed to a large extent by a wash with 5-10% ACN in water.…”

Section: Ah + Spa -----mentioning

confidence: 97%

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On-line derivatization of peptides for improved sequence analysis by micro-column liquid chromatography coupled with electrospray ionization-tandem mass spectrometry

Cárdenas

Heeft

Jong

1997

Rapid Commun. Mass Spectrom.

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An on-line method has been developed for the derivatization and coupled liquid chromatography (LC)/electrospray ionization (ESI) MS analysis of peptides at the femtomol level. Peptides are reacted with N-succinimidyl-2(3-pyridyl)acetate (SPA) in buffered aqueous medium at pH7 following loading on a precolumn (PC) in a microcolumn switching system. The fast-hydrolysing reagent is dissolved in dry methanol and mixed, in a 3 vol% ratio, with a buffer just before reaching the sample on the PC. Following the reaction and wash, the N-pyridylacetyl (PA) derivatives formed are transferred to the analytical micro-high-performance (HP) LC column for separation and subsequent ESI tandem MS analysis. The reaction is nearly quantitative and takes place selectively at the N-terminal and lysine amino functions, the latter providing a chemical means to distinguish between isobaric residues lysine and glutamine. In some cases, a minor reaction was observed with the tyrosine hydroxyl group. The N-terminal PA group was able to alter the collision-induced fragmentation pathway of peptides in favour of the formation of abundant b-type ions, a helpful feature for sequence elucidation of unknown peptides, particularly with quadrupole ion trap instruments.

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Section: Ah + Spa -----mentioning

confidence: 97%

“…The injection loop was a piece of fused-silica tubing with a volume of 40 µL. The precolumn (PC) and analytical column were both home made according to the procedure of Hunt et al 14 The dimensions of the PC were: 0.2 mm i.d. fused-silica filled with 4.5 cm Poros RP (10 µm particle size).…”

Section: Instrumentationmentioning

confidence: 99%

On-line derivatization of peptides for improved sequence analysis by micro-column liquid chromatography coupled with electrospray ionization-tandem mass spectrometry

Cárdenas

Heeft

Jong

1997

Rapid Commun. Mass Spectrom.

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“…Product ion mass spectra (see the review [40]) generated by the ion trap were searched automatically against existing database archives using the SEQUEST computer program [37,38], see Fig. (4), or interpreted manually using the de novo approach developed in Hunt's laboratory for low energy CID data [41,42]. The use of tandem mass spectrometry with peptides has as its great strength the fact that there are usually many redundant measurements for each protein to generate sufficient partial sequence information to match with a database, or alternatively, to develop the necessary primers to locate an unknown gene using the tools of molecular biology.…”

Section: D or Not 2d: Gel Analysis Hplc And Mass Spectrometrymentioning

confidence: 99%

Mass Spectrometry-Based Proteomics and its Application to Studies of Porphyromonas gingivalis Invasion and Pathogenicity

Lamont¹,

Meilă²,

Xia³

et al. 2006

IDDT

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Porphyromonas gingivalis is a Gram-negative anaerobe that populates the subgingival crevice of the mouth. It is known to undergo a transition from its commensal status in healthy individuals to a highly invasive intracellular pathogen in human patients suffering from periodontal disease, where it is often the dominant species of pathogenic bacteria. The application of mass spectrometry-based proteomics to the study of P. gingivalis interactions with model host cell systems, invasion and pathogenicity is reviewed. These studies have evolved from qualitative identifications of small numbers of secreted proteins, using traditional gel-based methods, to quantitative whole cell proteomic studies using multiple dimension capillary HPLC coupled with linear ion trap mass spectrometry. It has become possible to generate a differential readout of protein expression change over the entire P. gingivalis proteome, in a manner analogous to whole genome mRNA arrays. Different strategies have been employed for generating protein level expression ratios from mass spectrometry data, including stable isotope metabolic labeling and most recently, spectral counting methods. A global view of changes in protein modification status remains elusive due to the limitations of existing computational tools for database searching and data mining. Such a view would be desirable for purposes of making global assessments of changes in gene regulation in response to host interactions during the course of adhesion, invasion and internalization. With a complete data matrix consisting of changes in transcription, protein abundance and protein modification during the course of invasion, the search for new protein drug targets would benefit from a more comprehensive understanding of these processes than what could be achieved prior to the advent of systems biology. KeywordsPorphyromonas gingivalis; gingival epithelial cells; proteomics; posttranslational modification; protein expression; invasion; database search algorithm; review; MudPIT Diseases that originate at mucous membranes and involve constituents of the normal microbiota are often multifactorial in origin. Nowhere is this more apparent than in the human oral sub-gingival crevice. In this environment, hundreds of bacterial species interact with the multicompartmental periodontal tissues that provide supporting structures for the teeth. Under normal conditions, the host tolerates this microbial burden and the periodontal tissues remain healthy. However ecological shifts can occur that cause the microbiota to acquire a pathogenic *Address correspondence to this author at the Department of Chemical Engineering, Box 355014, University of Washington, Seattle, Washington 98195; Telephone: (206) 616 8071; E-mail mhackett@u.washington.edu. NIH Public Access Author ManuscriptInfect Disord Drug Targets. Author manuscript; available in PMC 2009 April 7. Published in final edited form as:Infect Disord Drug Targets. 2006 September ; 6(3): 311-325. NIH-PA Author ManuscriptNIH-PA Author Manuscript ...

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“…T he field of protein mass spectrometry has undergone a major paradigm shift over the past several years, moving from the analysis of individual proteins to the analysis of total digests of complex protein mixtures (also know as shotgun proteomics). The development of shotgun proteomics by Yates [1-3] couples nanoscale LC for peptide analysis [4,5], automated MS to MS/MS data acquisition software and hardware on modern tandem mass spectrometers [6], along with automated database searching software of modern search engines [7,8]. Shotgun proteomics has significantly increased the information content of protein mass spectrometry experiments, while at the same time significantly reducing the total analysis time required to analyze a complex protein mixture.…”

mentioning

confidence: 99%

“…The development of shotgun proteomics by Yates [1][2][3] couples nanoscale LC for peptide analysis [4,5], automated MS to MS/MS data acquisition software and hardware on modern tandem mass spectrometers [6], along with automated database searching software of modern search engines [7,8]. Shotgun proteomics has significantly increased the information content of protein mass spectrometry experiments, while at the same time significantly reducing the total analysis time required to analyze a complex protein mixture.…”

mentioning

confidence: 99%

A novel Interface for variable flow nanoscale LC/MS/MS for improved proteome coverage

Vissers¹,

Blackburn

Moseley

2002

J. Am. Soc. Mass Spectrom.

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A variable flow "peak trapping" liquid chromatography (LC) interface has been developed for the coupling of nanoscale LC to electrospray ionization mass spectrometry (ESI-MS). The presented peak trapping LC interface allows for the extended analysis time of co-eluting compounds and has been employed for the identification of proteins via tandem mass spectrometry (MS/MS). The variable flow process can be controlled either manually or in a completely automated manner where the mass spectrometer status determines the status of the variable flow interface. When the mass spectrometer operates in MS survey mode, the interface is operated in a so-called "high-flow" mode. Alternatively, the interface is operated in a "low-flow" mode during MS/MS analysis. In the "high-flow" mode of the variable flow process the column flow rate is typically around 200 nL/min, whereas in the "low-flow" mode the column effluent is introduced into the source of the mass spectrometer at 25 nL/min. In addition to the flow reduction during MS/MS analysis, the gradient is paused to preserve the peptide separation on the analytical nanoscale LC column. T he field of protein mass spectrometry has undergone a major paradigm shift over the past several years, moving from the analysis of individual proteins to the analysis of total digests of complex protein mixtures (also know as shotgun proteomics). The development of shotgun proteomics by Yates [1-3] couples nanoscale LC for peptide analysis [4,5], automated MS to MS/MS data acquisition software and hardware on modern tandem mass spectrometers [6], along with automated database searching software of modern search engines [7,8]. Shotgun proteomics has significantly increased the information content of protein mass spectrometry experiments, while at the same time significantly reducing the total analysis time required to analyze a complex protein mixture. Improvements in sample sensitivity and proteome coverage have been observed with this approach, compared to the "traditional" approach of using 2-D gels to separate the proteins, followed by in-gel digestion and analysis of the resulting peptides by mass spectrometry [3]. The shift in approach from the MS and MS/MS analysis of individual proteins to the analysis of complex mixtures of proteins has lead to the most significant figure of merit in proteomic analysis becoming the extent of proteome coverage obtained. Analytical methods that increase the coverage of the proteins in a sample will increase the impact of proteomics on biology.For a typical 50 kD protein, one would expect to have on the order of 40 -50 tryptic peptides. Characterization of the complex mixture of peptides resulting from a total digest of a complex protein mixture requires the mass spectrometric analysis of hundreds to many thousands of peptides. Clearly, the MS and MS/MS analysis of a sample comprised of hundreds to thousands of proteins is challenging. Modern mass spectrometers with data dependent scanning software are capable of acquiring MS and MS/MS data from many hund...

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Protein and Peptide Sequence Analysis by Tandem Mass Spectrometry in Combination with Either Capillary Electrophoresis or Micro-Capillary HPLC

Cited by 14 publications

References 11 publications

On-line derivatization of peptides for improved sequence analysis by micro-column liquid chromatography coupled with electrospray ionization-tandem mass spectrometry

On-line derivatization of peptides for improved sequence analysis by micro-column liquid chromatography coupled with electrospray ionization-tandem mass spectrometry

Mass Spectrometry-Based Proteomics and its Application to Studies of Porphyromonas gingivalis Invasion and Pathogenicity

A novel Interface for variable flow nanoscale LC/MS/MS for improved proteome coverage

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