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Bioinformatics for Comparative Proteomics

Volume 694 of the series Methods in Molecular Biology pp 291-304

Date:

An Integrated Top-Down and Bottom-Up Strategy for Characterization of Protein Isoforms and Modifications

  • Si WuAffiliated withPacific Northwest National Laboratory
  • , Nikola TolićAffiliated withPacific Northwest National Laboratory
  • , Zhixin TianAffiliated withPacific Northwest National Laboratory
  • , Errol W. RobinsonAffiliated withPacific Northwest National Laboratory
  • , Ljiljana Paša-TolićAffiliated withPacific Northwest National Laboratory Email author 

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Abstract

Bottom-up and top-down strategies are two commonly used methods for mass spectrometry (MS) based protein identification; each method has its own advantages and disadvantages. In this chapter, we describe an integrated top-down and bottom-up approach facilitated by concurrent liquid chromatography-mass spectrometry (LC-MS) analysis and fraction collection for comprehensive high-throughput intact protein profiling. The approach employs a high resolution reversed phase (RP) LC separation coupled with LC eluent fraction collection and concurrent on-line MS with a high field (12 T) Fourier-transform ion cyclotron resonance (FTICR) mass spectrometer. Protein elusion profiles and tentative modified protein identification are made using detected intact protein mass in conjunction with bottom-up protein identifications from the enzymatic digestion and analysis of corresponding LC fractions. Specific proteins of biological interest are incorporated into a target ion list for subsequent off-line gas-phase fragmentation that uses an aliquot of the original collected LC fraction, an aliquot of which was also used for bottom-up analysis.

Key words

Protein Peptide Proteomics Mass spectrometry LC-MS Top-down Bottom-up FT-ICR MS FTMS Post-translational modification PTM