Enhanced protein identification using graphite-modified MALDI plates for offline LC-MALDI-MS/MS bottom-up proteomics

Abstract

The use of offline liquid chromatography-matrix assisted laser desorption/ionization (LC-MALDI) tandem mass spectrometry (MS/MS) for bottom-up proteomics offers advantages in terms of cost, ease of use, and the time-decoupled nature of the separation step and the mass analysis. A method was developed to improve the capabilities of LC-MALDI-MS/MS in terms of protein identification in a bottom-up proteomic workflow. Enhanced protein identification is achieved by an increase in the MALDI signal intensity of the precursor peptides brought about by coating the MALDI plate with a thin film of graphite powder. Using the Escherichia coli proteome, it is demonstrated that the graphite-modified MALDI plates used in an offline LC-MALDI-MS/MS bottom-up protocol led to a 50-135% increase in the number of peptide identifications, and a concomitant 21%-105% increase in the number of proteins inferred. We identify factors that lead to improvements in peptide sequence identifications and in the number of unique proteins identified when compared to using an unmodified MALDI plate. These improvements are achieved using a low cost approach that it is easy to implement, requires no hardware/protocol modification, it is compatible with LC and adds no additional analysis time.

Keywords: Bottom-up proteomics; MALDI-TOF/TOF; Offline LC-MALDI; Protein identification.