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.

Chemically heterogeneous carbon dots enhanced cholesterol detection by MALDI TOF mass spectrometry

“…Various organic compounds have been used as matrices. Examples are 1) aromatic compounds that absorb in the laser emission region, such as benzoic acid or cinnamic acid derivatives, 2) liquid crystals, such as organic ammonium salts (butyl ammonium salts), which are used for a soft ionization of labile analytes [4,5] and 3) inorganic materials such as graphite or metal-oxide nanoparticles [6,7]. The matrix should be selected based on a high signal-to-noise (S/N) ratio of an analyte signal.…”

Detection and identification of a protein biomarker in antibiotic‐resistant Escherichia coli using intact protein LC offline MALDI‐MS and MS/MS

“…Digested samples were then analyzed by offline LC MALDI‐ToF‐ToF‐MS/MS using a MALDI plate precoated with graphite powder to increase the peptide signal intensity and thus the number of protein identifications (Maus et al ).…”

Carbon-Based Nanoparticles and Their Surface-Modified Counterparts as MALDI Matrices

“…Ease of analysis is another prominent advantage of using graphite as it reduces the need for tedious analyte extraction steps before analysis, as in some way “extraction” and matrix application is performed at the same time. The target molecules, adsorbed via van der Waals interactions, on the surface of graphite particles can be transferred directly to the MALDI plate, or alternatively, the sample can be deposited onto the graphite-coated target plate. − …”