Isobaric Peptide Termini Labeling for MS/MS-Based Quantitative Proteomics

The competence-stimulating peptide (CSP) and the sigX-inducing peptide (XIP) are known to induce Streptococcus mutans competence for genetic transformation. For both pheromones, direct identification of the native peptides has not been accomplished. The fact that extracellular XIP activity was recently observed in a chemically defined medium devoid of peptides, as mentioned in an accompanying paper (K. Desai, L. Mashburn-Warren, M. J. Federle, and D. A. Morrison, J. Bacteriol. 194:3774 -3780, 2012), provided ideal conditions for native XIP identification. To search for the XIP identity, culture supernatants were filtered to select for peptides of less than 3 kDa, followed by C 18 extraction. One peptide, not detected in the supernatant of a comS deletion mutant, was identified by tandem mass spectrometry (MS/MS) fragmentation as identical to the ComS C-terminal sequence GLDWWSL. ComS processing did not require Eep, a peptidase involved in processing or import of bacterial small hydrophobic peptides, since eep deletion had no inhibitory effect on XIP production or on synthetic XIP response. We investigated whether extracellular CSP was also produced. A reporter assay for CSP activity detection, as well as MS analysis of supernatants, revealed that CSP was not present at detectable levels. In addition, a mutant with deletion of the CSP-encoding gene comC produced endogenous XIP levels similar to those of a nondeletion mutant. The results indicate that XIP pheromone production is a natural phenomenon that may occur in the absence of natural CSP pheromone activity and that the heptapeptide GLDWWSL is an extracellular processed form of ComS, possibly the active XIP pheromone. This is the first report of direct identification of a ComR/ComS pheromone.

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“…For accurate mass measurements, the lock mass option was enabled in MS mode. Other instrument parameters were set as previously described (18).…”

Section: Methodsmentioning

confidence: 99%

Extracellular Identification of a Processed Type II ComR/ComS Pheromone of Streptococcus mutans

et al. 2012

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“…Consequently, MS-based quantification techniques have been applied to proteomics data sets, including e.g. stable isotope labeling of amino acids in cell culture (SILAC) 1 (11), tandem mass tagging TMT (12), isobaric tags for relative and absolute quantification ITRAQ (13), and isobaric peptide termini labeling IPTL (14). SILAC enables peptides derived from different physiological conditions to be quantified at the MS level, whereas tandem mass tagging, isobaric tags for relative and absolute quantification, and isobaric peptide termini labeling yield isobaric peptides and thus require MS/MS data to reveal quantitative information for the peptides.…”

mentioning

confidence: 99%

ApoptoProteomics, an Integrated Database for Analysis of Proteomics Data Obtained from Apoptotic Cells

Arntzen

Thiede

2012

Molecular & Cellular Proteomics

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Apoptosis is the most commonly described form of programmed cell death, and dysfunction is implicated in a large number of human diseases. Many quantitative proteome analyses of apoptosis have been performed to gain insight in proteins involved in the process. This resulted in large and complex data sets that are difficult to evaluate. Therefore, we developed the ApoptoProteomics database for storage, browsing, and analysis of the outcome of large scale proteome analyses of apoptosis derived from human, mouse, and rat. The proteomics data of 52 publications were integrated and unified with protein annotations from UniProt-KB, the caspase substrate database homepage (CASBAH), and gene ontology. Currently, more than 2300 records of more than 1500 unique proteins were included, covering a large proportion of the core signaling pathways of apoptosis. Analysis of the data set revealed a high level of agreement between the reported changes in directionality reported in proteomics studies and expected apoptosisrelated function and may disclose proteins without a current recognized involvement in apoptosis based on gene ontology. Comparison between induction of apoptosis by the intrinsic and the extrinsic apoptotic signaling pathway revealed slight differences. Furthermore, proteomics has significantly contributed to the field of apoptosis in identifying hundreds of caspase substrates. The database is available at http

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“…31,36 We also used quantitative proteomics to assess whether the transcriptomic signatures were reflected at the level of the proteome. We used a newly developed technique called isobaric peptide terminal labeling (IPTL) technology, 52 which can be used to label proteins in freshly prepared extracts from whole worms 36 or from isolated tissues like the germline. 53 It is a very good alternative for C. elegans proteomic studies where metabolic labeling has some limitations.…”

Section: Hormesis Maintains Wildtype Phenotypes In Dna Repair Mutantsmentioning

confidence: 99%

Active transcriptomic and proteomic reprogramming in theC. elegansnucleotide excision repair mutant xpa-1

Kassahun

Nilsen

2013

Worm

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O xidative stress promotes human aging and contributes to common neurodegenerative diseases. Endogenous DNA damage induced by oxidative stress is believed to be an important promoter of neurodegenerative diseases. Although a large amount of evidence correlates a reduced DNA repair capacity with aging and neurodegenerative disease, there is little direct evidence of causality. Moreover, the contribution of oxidative DNA damage to the aging process is poorly understood. We have used the nematode Caenorhabditis elegans to study the contribution of oxidative DNA damage and repair to aging. C. elegans is particularly well suited to tackle this problem because it has a minimum complexity DNA repair system, which enables us to circumvent the important limitation presented by the extensive redundancy of DNA repair enzymes in mammals. Oxidative DNA Damage and Aging in C. elegansThe free radical theory of aging is an example of a hypothesis that, because it intuitively makes sense, is attractive both to scientists and to the general public. The theory has been enormously influential and has inspired scientists to test its implications ever since its first formulation by Denham Harman. 1 As such it is a good theory because it suggests many testable hypotheses. However, few experiments have provided direct support of the original theory. This has led to the formulation of many offshoots like the mitochondrial theory of aging and the stochastic damage accumulation theory of aging.One prediction that can be made from all of these theories is that oxidative damage to cellular macromolecules contributes to the progressive loss of function associated with aging. As DNA, unlike other cellular macromolecules, cannot be replaced in its entirety, it seems logical that maintenance of genomic stability would promote longevity and limit functional loss during aging.A role for passive and stochastic accumulation of oxidative DNA damage in aging is often dismissed by scientists outside the DNA repair field for three principal reasons; first, even though levels of reactive oxygen species (ROS) increase with age 2,3 and DNA repair capacity diminishes with age, 4 it has been difficult to unequivocally show that oxidative DNA damage accumulates with age. Also, since a plethora of different types of DNA lesions are induced by ROS, it is not entirely clear which types of lesions are more relevant to the aging phenotype. Second, there is no good correlation between mutation accumulation and aging. [5][6][7] Third, mutants that fail to repair oxidative DNA damage show normal lifespan. However, there are technical and biological factors that would explain these observations and further research is needed to determine whether and how oxidative DNA damage contributes to aging. Segmental Progeroid NER SyndromesIt is often overlooked that DNA damage may not only be mutagenic, but also

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Isobaric Peptide Termini Labeling for MS/MS-Based Quantitative Proteomics

Cited by 103 publications

References 48 publications

Extracellular Identification of a Processed Type II ComR/ComS Pheromone of Streptococcus mutans

Extracellular Identification of a Processed Type II ComR/ComS Pheromone of Streptococcus mutans

ApoptoProteomics, an Integrated Database for Analysis of Proteomics Data Obtained from Apoptotic Cells

Active transcriptomic and proteomic reprogramming in theC. elegansnucleotide excision repair mutant xpa-1

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