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Comprehensive analysis of proteins of pH fractionated samples using monolithic LC/MS/MS, intact MW measurement and MALDI-QIT-TOF MS.

ABSTRACT: A comprehensive platform that integrates information from the protein and peptide levels by combining various MS techniques has been employed for the analysis of proteins in fully malignant human breast cancer cells. The cell lysates were subjected to chromatofocusing fractionation, followed by tryptic digestion of pH fractions for on-line monolithic RP-HPLC interfaced with linear ion trap MS analysis for rapid protein identification. This unique approach of direct analysis of pH fractions resulted in the identification of large numbers of proteins from several selected pH fractions, in which approximately 1.5 microg of each of the pH fraction digests was consumed for an analysis time of ca 50 min. In order to combine valuable information retained at the protein level with the protein identifications obtained from the peptide level information, the same pH fraction was analyzed using nonporous (NPS)-RP-HPLC/ESI-TOF MS to obtain intact protein MW measurements. In order to further validate the protein identification procedures from the fraction digest analysis, NPS-RP-HPLC separation was performed for off-line protein collection to closely examine each protein using MALDI-TOF MS and MALDI-quadrupole ion trap (QIT)-TOF MS, and excellent agreement of protein identifications was consistently observed. It was also observed that the comparison to intact MW and other MS information was particularly useful for analyzing proteins whose identifications were suggested by one sequenced peptide from fraction digest analysis.

SUBMITTER: Yoo C

PROVIDER: S-EPMC3426914 | biostudies-literature | 2007 Mar

REPOSITORIES: biostudies-literature

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Comprehensive analysis of proteins of pH fractionated samples using monolithic LC/MS/MS, intact MW measurement and MALDI-QIT-TOF MS.

Yoo Chul C Patwa Tasneem H TH Kreunin Paweena P Miller Fred R FR Huber Christian G CG Nesvizhskii Alexey I AI Lubman David M DM

Journal of mass spectrometry : JMS 20070301 3

A comprehensive platform that integrates information from the protein and peptide levels by combining various MS techniques has been employed for the analysis of proteins in fully malignant human breast cancer cells. The cell lysates were subjected to chromatofocusing fractionation, followed by tryptic digestion of pH fractions for on-line monolithic RP-HPLC interfaced with linear ion trap MS analysis for rapid protein identification. This unique approach of direct analysis of pH fractions resul ...[more]

PMID: 17206599

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Project description:Mitral valve disease (MVD) is an important and most frequently acquired heart disease found in dogs. MVD is classified into different stages according to its severity. There is a challenge in differentiation between asymptomatic and symptomatic stages of the MVD. Moreover, pulmonary hypertension (PH) is a common complication in dogs affected by MVD. In clinical practice, there are also some limitations to identify PH. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a technique that can characterize specific patterns of peptide mass called peptide barcodes from various samples. Besides, in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS), potential peptide sequences associated with specific conditions could be identified. The present study aimed to use MALDI-TOF coupled with LC-MS/MS to characterize specific peptide barcodes and potential peptide candidates in serum samples from healthy dogs, dogs with MVD stage B (MVD B, asymptomatic stage), MVD stage C (MVD C, symptomatic stage), MVD stage B with PH (MVD B PH), and MVD stage C with PH (MVD C PH). Discrete clusters of the 5 sample groups were identified by 3D plot analysis. Peptide barcodes also revealed differences in peptide patterns among the 5 groups. Six amino acid sequences of peptide candidates at 1,225.60, 1,363.85, 1,688.71, 1789.52, 2020.21, and 2156.42 Da were identified as part of the proteins CLCN1, CLUL1, EDNRA, PTEN, SLC39A7, and CLN6, respectively. The network interactions between these discovered proteins and common cardiovascular drugs were also investigated. These results demonstrate that MALDI-TOF MS has promise as an optional technique for diagnosing dogs affected by asymptomatic and symptomatic stages of MVD with and without PH. Further studies are required to identify peptide barcodes in dogs with other diseases to create peptide barcode databases in veterinary medicine before using this method as a novel diagnostic tool in the future.

Dataset Information

Comprehensive analysis of proteins of pH fractionated samples using monolithic LC/MS/MS, intact MW measurement and MALDI-QIT-TOF MS.

Publications

Comprehensive analysis of proteins of pH fractionated samples using monolithic LC/MS/MS, intact MW measurement and MALDI-QIT-TOF MS.

Similar Datasets

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