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LC-MALDI and MALDI-MS/MS for the Analysis of Complex Peptide Mixtures

Joseph Fox1, Joanne B. Connolly2, Matthew E. Openshaw2
1Shimadzu Scientifi c Inc., Columbia, US; 2Shimadzu Biotech/Kratos, UK


Introduction
LC-MALDI provides several advantages over conventional single spot (i.e. no separation) acquisitions for the analysis of complex peptide mixtures. These include (i) concentration and separation of the peptides prior to analysis, (ii) extended time available for MS/MS interrogation, (iii) separation of isobaric peptides resulting in increased sequence coverage. The ability to separate and concentrate the peptides prior to analysis results in higher sensitivity and means that more MS/MS spectra can be generated from the sample. Rather than acquiring all the MS/MS spectra from the same spot, with the sample being depleted with each MS/MS spectrum acquired, MS/MS spectra are generated from different positions on the MALDI target and sample depletion is less of an issue.

In the LC-MALDI technique, the protein mixture is enzymatically digested and the resulting peptides are separated prior to MALDI analysis (see Figure 1). Automated MALDI time-of-fl ight (TOF) mass spectrometric (MS) analysis is used to select suitable candidates for subsequent MS/MS fragmentation and proteins are identifi ed using the MS/MS data obtained for the individual peptides. We present results obtained using a new MALDI-TOF mass spectrometer, capable of true high-energy MS/MS (AXIMA-TOF2), demonstrating the utility of this technique for the analysis of complex protein mixtures. Also presented is data obtained for an iTRAQ labelled protein mixture, including a novel MALDI-MS/MS analysis mode (Low Mass Zoom (LMZ)) which can be used to enhance fragment ion resolution for low mass fragment ions, e.g. diagnostic ions used for relative quantifi cation (iTRAQ
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