Enhancement in MALDI-TOF MS analysis of the low molecular weight human serum proteome

The combination in advances in MALDI-TOF MS instrumentation(1) and serum sample preparation techniques,(2-4) has lead to the emergence of MALDI serum protein expression profiling as a promising tool for biomarker discovery.(5) However, three factors still pose significant challenges for MALDI profiling of serum: the limited mass window of MALDI, serum protein complexity,(6) and analytical reproducibility.(7, 8) MALDI has a small mass preference due to the ionization efficiency of intact protein molecules and the focusing of flight of ions towards the detector. In linear mode, TOF analyzers have limited sensitivity for masses above 20 kDa. To address this, we previously reported the enhancement of MALDI broad mass range detection of protein signals.(9) Spanning a 100 kDa mass range, we improved the sensitivity of detection by an order of magnitude through the combined optimization of sample preparation, instrument parameters and data processing procedures. The complexity of the blood proteome is very high, with protein concentrations differing by up to ten orders of magnitude.(10) This large dynamic range exceeds current proteomic analytical capabilities; thus analysis of easily prepared subproteomes of serum or plasma is essential. Here, we shift our focus back to the enhancement of MALDI analysis in the low mass regime. The low molecular weight (LMW) subcomponent of serum promises to be a rich source of undiscovered biomarkers, as biological processes give rise to a plethora of proteolytic protein fragments.(11) Currently, there is no consensus on what constitutes the mass limits of this derivative proteome (also termed peptidome). However, “<15 kDa” is often cited in the literature based on a serum MALDI study using 20 MWCO filters.(12) To date, small native protein/peptide mass measurements have been mainly conducted in reflectron mode (< 4 kDa),(13) and linear mode up to 10 kDa.(14) However, no systematic comparison has been described for a combination of preparation steps: spotting, filtering and matrix choice, to optimize the performance. A rigorous reproducibility study for different preparation strategies is likewise missing, which prevents extension of the technique to clinical proteomics applications. The purpose of this work was to provide such a systematic comparison, using rigorous performance metrics to optimize sample preparation for LMW serum proteome profiling by MALDI-TOF MS analysis up to 20 kDa. We explored a combination of MALDI sample preparation and spotting methods. Procedures that gave the best results included: 1) MALDI spotting with a thin layer technique using sinapinic acid on a ground steel plate, and 2) centrifugal ultrafiltration with 50000 MWCO filters in the presence of 2% TFA, followed by desalting with C3 magnetic beads. Ultrafiltration has been utilized previously to improve MALDI profiling of serum.(2, 13, 15-17) However, our approach extensively improves MALDI peak intensities of higher MW peptides and small proteins, thus increasing biomarker coverage in the 3-20 kDa range. Reproducibility studies of a protein standard and serum samples gave excellent results, all of which suggests that this procedure can be a useful tool for proteome profiling of the LMW fraction of serum.