Efficient High-Throughput Discovery of Large Peptidic Hormones and Biomarkers

Taylor, Steven W.; Andon, Nancy L.; Bilakovics, James M; Lowe, Carolyn; Hanley, Michael R.; Pittner, Richard A.; Ghosh, Soumitra S.

doi:10.1021/pr0600982

Cited by 20 publications

(36 citation statements)

References 30 publications

(49 reference statements)

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“…A few laboratories have started to acquire large-scale MS/MS data in addition to MS data with the ultrahigh mass accuracy afforded by FTMS (42,43). This simple switch in collecting both intact MS and MS/MS fragmentation data in the FTMS has not been compared directly with the more widespread mode of ''FT/ion trap'' data acquisition-or with TOF instruments.…”

Section: Winds Of Change: High-resolution Tandem Mass Spectrometrymentioning

confidence: 99%

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Precision proteomics: The case for high resolution and high mass accuracy

Mann

Kelleher

2008

Proc. Natl. Acad. Sci. U.S.A.

398

353

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Proteomics has progressed radically in the last 5 years and is now on par with most genomic technologies in throughput and comprehensiveness. Analyzing peptide mixtures by liquid chromatography coupled to high-resolution mass spectrometry (LC-MS) has emerged as the main technology for in-depth proteome analysis whereas two-dimensional gel electrophoresis, low-resolution MALDI, and protein arrays are playing niche roles. MS-based proteomics is rapidly becoming quantitative through both label-free and stable isotope labeling technologies. The latest generation of mass spectrometers combines extremely high resolving power, mass accuracy, and very high sequencing speed in routine proteomic applications. Peptide fragmentation is mostly performed in lowresolution but very sensitive and fast linear ion traps. However, alternative fragmentation methods and high-resolution fragment analysis are becoming much more practical. Recent advances in computational proteomics are removing the data analysis bottleneck. Thus, in a few specialized laboratories, ''precision proteomics'' can now identify and quantify almost all fragmented peptide peaks. Huge challenges and opportunities remain in technology development for proteomics; thus, this is not ''the beginning of the end'' but surely ''the end of the beginning.''

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Section: Winds Of Change: High-resolution Tandem Mass Spectrometrymentioning

confidence: 99%

“…3 A vs. B). Here, peptides longer than Ϸ20 residues (48,49) or large endogenous peptides (42) are sought for analysis to increase the sequence coverage provided by each successfully identified peptide.…”

Section: Sequencing Larger Peptides and Top-down Proteomicsmentioning

confidence: 99%

Precision proteomics: The case for high resolution and high mass accuracy

Mann

Kelleher

2008

Proc. Natl. Acad. Sci. U.S.A.

398

353

View full text Add to dashboard Cite

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“…The direct tissue and single neuron analysis by MALDI MS has enabled the discovery of hundreds of neuropeptides in the last decade, and the neuronal homogenate analysis by fractionation and subsequent ESI or MALDI MS has yielded an equivalent number of new brain peptides (5). Several recent peptidome studies, including the work by Dowell et al (10), have used the specificity of FTMS for peptide discovery (10,(13)(14)(15). Here, we combine the ability to fragment ions at ultrahigh mass accuracy (16) with a software pipeline designed for neuropeptide discovery.…”

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confidence: 99%

Endogenous Peptide Discovery of the Rat Circadian Clock

Lee

Atkins

Hatcher

et al. 2010

Molecular & Cellular Proteomics

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Understanding how a small brain region, the suprachiasmatic nucleus (SCN), can synchronize the body's circadian rhythms is an ongoing research area. This important time-keeping system requires a complex suite of peptide hormones and transmitters that remain incompletely characterized. Here, capillary liquid chromatography and FTMS have been coupled with tailored software for the analysis of endogenous peptides present in the SCN of the rat brain. After ex vivo processing of brain slices, peptide extraction, identification, and characterization from tandem FTMS data with <5-ppm mass accuracy produced a hyperconfident list of 102 endogenous peptides, including 33 previously unidentified peptides, and 12 peptides that were post-translationally modified with amidation, phosphorylation, pyroglutamylation, or acetylation. This characterization of endogenous peptides from the SCN will aid in understanding the molecular mechanisms that mediate rhythmic behaviors in mammals. Molecular & Cellular Proteomics 9:285-297, 2010.

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“…In their experiments they saw enrichment on the order of 10 3 for the peptides of interest. Many groups have enriched small proteins and polypeptides in the 20 -60-amino acid range with size exclusion chromatography or reverse phase chromatography (25)(26)(27). The authors were able to analyze the sample without enzymatic digestion because of the size of the polypeptides.…”

mentioning

confidence: 99%

Use of an Immunoaffinity-Mass Spectrometry-based Approach for the Quantification of Protein Biomarkers from Serum Samples of Lung Cancer Patients

Nicol¹,

Han²,

Kim³

et al. 2008

Molecular & Cellular Proteomics

106

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It is a challenging task to verify and quantify potential biomarkers expressed at elevated levels in sera from cancer patients. An immunoaffinity-mass spectrometrybased approach has been developed using antibodies to enrich proteins of interest from sera followed by mass spectrometry-based quantification. Antibodies specific to the protein of interest were immobilized to hydrazide resin via the carbohydrate moiety on the Fc region of the antibody. Captured proteins were eluted, reduced, alkylated, and digested with trypsin. Peptides were analyzed by LC coupled with multiple reaction monitoring approach, and quantification was achieved by the addition of stable isotope-labeled (heavy) standard peptides. Using this methodology, we were able to achieve a linear response from 15 to 250 ng/ml for carcinoembryonic antigen (CEA), a known tumor biomarker. Moreover we observed elevated levels of CEA in sera samples from lung cancer patients that to our knowledge is the first time that circulating CEA has been detected by mass spectrometry-based analysis. This approach was further applied to potential protein biomarkers discovered from tumor cell lines and tumor tissues. A linear response was obtained from a multiplex spiking experiment in normal human sera for secretory leukocyte peptidase inhibitor (4 -500 ng/ml), tissue factor pathway inhibitor (TFPI) (42-1000 ng/ml), tissue factor pathway inhibitor 2 (TFPI2) (2-250 ng/ml), and metalloproteinase inhibitor 1 (TIMP1) (430 -1000 ng/ml). A replicate experiment for a single concentration value yielded a relative coefficient of variation better than 11% for TFPI, secretory leukocyte peptidase inhibitor, and TFPI2. The expression level of the proteins in lung cancer patient sera was assayed by an immunoaffinitymultiple reaction monitoring method, and the results were comparable with those obtained from ELISA. This immunoaffinity-mass spectrometry-based quantification approach thus provides a specific and accurate assay for verifying the expression of potential biomarkers in patient serum samples especially for those proteins Biomarkers have attracted significant attention for disease diagnosis, prognosis, and therapeutic response monitoring in recent years (1). The development of clinical biomarkers involves discovery, verification, and validation. Progress in genomics and proteomics has led to a large number of potential biomarkers (2-4). However, the promise of these discoveries requires proper verification and validation in the disease of interest (5). The ELISA has been the established platform for quantification of proteins in sera. However, for many of these newly identified potential biomarkers there are no ELISA kits available, and development of an ELISA kit is time-consuming (6), making it impractical to examine large panels of biomarkers. Although ELISA remains the "gold standard" for clinical applications, mass spectrometry-based quantification analysis has been shown as a possible intermediate technology in the validation process to prioritize candidates for ELIS...

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Efficient High-Throughput Discovery of Large Peptidic Hormones and Biomarkers

Cited by 20 publications

References 30 publications

Precision proteomics: The case for high resolution and high mass accuracy

Precision proteomics: The case for high resolution and high mass accuracy

Endogenous Peptide Discovery of the Rat Circadian Clock

Use of an Immunoaffinity-Mass Spectrometry-based Approach for the Quantification of Protein Biomarkers from Serum Samples of Lung Cancer Patients

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