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用LTQ-Orbitrap对小分子血浆蛋白进行自上而下分析,转甲状腺素蛋白和血红蛋白质谱临床分析的潜力

发布时间: 2014-03-11 13:58 来源:华质泰科生物技术(北京)有限公司
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Int J Mass Spectrom. 2011 Mar 1;300(2-3):130-142.

Top-Down Analysis of Small Plasma Proteins Using an LTQ-Orbitrap. Potential for Mass Spectrometry-Based Clinical Assays for Transthyretin and Hemoglobin.

Abstract

Transthyretin (TTR) amyloidosis and hemoglobinopathies are the archetypes of molecular diseases where point mutation characterization is diagnostically critical. We have developed a Top-down analytical platform for variant and/or modified protein sequencing and are examining the feasibility of using this platform for the analysis of hemoglobin/TTR patient samples and evaluating the potential clinical applications. The platform is based on a commercial high resolution hybrid orbitrap mass spectrometer (LTQ-Orbitrap(™)) with automated sample introduction; automated data analysis is performed by our own software algorithm (BUPID topdown).The analytical strategy consists of iterative data capture, first recording a mass profile of the protein(s). The presence of a variant is revealed by a mass shift consistent with the amino acid substitution. Nozzle-skimmer dissociation (NSD) of the protein(s) yields a wide variety of sequence-defining fragment ions. The fragment ion containing the amino acid substitution or modification can be identified by searching for a peak exhibiting the mass shift observed in the protein mass profile. This fragment ion can then be selected for MS/MS analysis in the ion trap to yield sequence information permitting the identification of the variant. Substantial sequence coverage has been obtained in this manner. This strategy allows for a stepwise MS/MS analysis of the protein structure. The sequence information obtained can be supplemented with whole protein NSD fragmentation and MS/MS analysis of specific protein charge states. The analyses of variant forms of TTR and hemoglobin are presented to illustrate the potential of the method.

The samples were introduced into a “Classic” LTQ-Orbitrap mass spectrometer (Thermo-Fisher Corp., Waltham, MA) using a TriVersa
NanoMate  system  (Advion  Biosciences,  Ithaca,  NY). 

 

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