Defining Absolute Confidence Limits in the Identification of <i>Caulobacter</i> Proteins by Peptide Mass Mapping

Karty, Jonathan A.; Ireland, Marcia M. E.; Brun, Yves V.; Reilly, James P.

doi:10.1021/pr025518b

Cited by 25 publications

(35 citation statements)

References 48 publications

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“…Spot analysis was performed as described previously (Karty et al , 2002b). Briefly, spots were cut from the second‐dimension gels using cut‐off P200 pipette tips.…”

Section: Methodsmentioning

confidence: 99%

Proteomic analysis of the Caulobacter crescentus stalk indicates competence for nutrient uptake

Ireland¹,

Karty

Quardokus³

et al. 2002

Molecular Microbiology

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Summary Caulobacter crescentus , a Gram‐negative α ‐purple proteobacterium, is an oligotroph that lives in aquatic environments dilute in nutrients. This bacterium divides asymmetrically. Part of this asymmetric cell division involves the formation of a prosthecum at one pole, referred to as the stalk, which replaces the flagellum of the motile swarmer cell. Little is known about the synthesis or function of the stalk. The stalk is an extension of the cell membranes and peptidoglycan layer, and stalk elongation is stimulated by phosphate starvation. In this study, we have taken advantage of two‐dimensional gel (2D gel) electro‐phoresis as well as the fully sequenced genome of Caulobacter to study the proteome of the stalk. We modified a stalk‐shedding mutant strain of Caulobacter crescentus to increase the yield of stalk material shed and performed 2D gel electrophoresis of purified stalks and cellular fractions. Comparison of the stalk 2D gel with the 2D gels of cell membrane and soluble fractions showed that the stalk is mostly free of cytoplasmic proteins and has a profile very similar to that of the cell membrane. Of the 172 proteins on a stalk 2D gel, we report the identification of 64 spots, corresponding to 39 different proteins present in the stalk of Caulobacter. The identifications include several TonB‐dependent receptors, two OmpA family proteins, a dipeptidase, GlpQ, two alkaline phosphatases, 3‐phytase, a putative TolC protein and 11 proteins of unknown function. These identifications are consistent with the hypothesis that the stalk plays a role in nutrient uptake.

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“…Spot analysis was performed as described previously (Karty et al , 2002b). Briefly, spots were cut from the second‐dimension gels using cut‐off P200 pipette tips.…”

Section: Methodsmentioning

confidence: 99%

Proteomic analysis of the Caulobacter crescentus stalk indicates competence for nutrient uptake

Ireland¹,

Karty

Quardokus³

et al. 2002

Molecular Microbiology

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“…The identification of the N-terminal residue provides a database searching constraint. We simulated the application of this constraint using the translated genome of Caulobacter crescentus and a database analysis program (PRODIGIES) that was written in house [34,35]. When candidate sequences are limited by both the mass of a precursor ion and the identity of the N-terminal residue, the number of candidate sequences is reduced by approximately one order of magnitude.…”

Section: Fragmentation Of Electrosprayed Amidine Labeled Peptidesmentioning

confidence: 99%

Fragmentation of amidinated peptide ions

Beardsley

Reilly

2004

J. Am. Soc. Mass Spectrom.

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The collision-induced dissociation characteristics of amidinated and unmodified tryptic peptides are compared using an ion trap mass spectrometer with both electrospray ionization and matrix-assisted laser/desorption ionization (MALDI). Several fragmentation pathways in a number of tryptic peptides of various precursor charge states are found to be enhanced. The additional information conveyed by the observed fragment ions should facilitate protein identifications. (J Am Soc Mass Spectrom 2004, 15, 158Ϫ167)

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“…This phenomenon led to the development of signal-amplified tags (Cagney and Emili, 2002;Karty et al, 2002;Reilly, 2002, 2003). A mass-coded abundance tagging (MCAT) method was reported which involved the derivatization of the ε-amino groups of Lys residues using Omethylisourea; this labeling process causes a 42-Da mass difference (Cagney and Emili, 2002), as indicated in Fig.…”

Section: Guanidination and Gunidination-like Labelingmentioning

confidence: 99%

“…The guanidination (or guanidination-like modification) does indeed improve the ionization of peptides and the signals of y ions, which could be used for de novo sequencing. In fact, 30% more proteins were identified from the Caulobacter proteome after guanidination (Karty et al, 2002). However, this MCAT (or QUEST) method possessed two major drawbacks for the large-scale analysis of protein expression.…”

Section: Guanidination and Gunidination-like Labelingmentioning

confidence: 99%

Recent Progress in Quantitative Proteomics Using Stable Isotope Labeling, Multidimensional Liquid Chromatography and Mass Spectrometry

Hsu¹,

Chen

2005

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Stable isotope labeling coupled with multidimensional liquid chromatography/tandem mass spectrometry is an advanced platform for global proteome-wide quantification. Compared to conventional methods, such as two dimensional polyacrylamide gel or electroblot based quantification methods, stable isotope labeling holds greater promise for accurate and large-scale quantitative analyses. Various labeling strategies have been developed to analyze protein expression, posttranslational modification, and protein/protein interactions, as well as for absolute quantification. There are advantages and disadvantages inherent with each method, but their applicability is dependent on many factors. In addition to the choice of which labeling chemistry to be used, there are several bottleneck issues associated with this approach that are of critical importance. These include finding effective multidimensional separation steps to resolve low abundant proteins present in relatively complicated mixtures, validating the methods, and interpreting the large amount of statistical data generated. This review covers the current progress in solving these concerns and summarizes the various labeling strategies and applications. We believe that a judicious integration of each component of the technique is crucial for the success of such a global systems approach. Based on the current progress, it is clear that the stable isotope labeling/mass spectrometry technique will find tremendous use in many fields, such as drug discovery, clinical diagnostics, disease prevention, basic biological research, and biotechnology.

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Defining Absolute Confidence Limits in the Identification of Caulobacter Proteins by Peptide Mass Mapping

Cited by 25 publications

References 48 publications

Proteomic analysis of the Caulobacter crescentus stalk indicates competence for nutrient uptake

Proteomic analysis of the Caulobacter crescentus stalk indicates competence for nutrient uptake

Fragmentation of amidinated peptide ions

Recent Progress in Quantitative Proteomics Using Stable Isotope Labeling, Multidimensional Liquid Chromatography and Mass Spectrometry

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