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

Abstract

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 * Correspondence to: Dr. Tina Gatlin, National Human Genome Research Institute, National Institutes of Health, Rockville, MD 20892 USA. christine.gatlin@nih.gov (current address). 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...

Prognostic accuracy of MALDI-TOF mass spectrometric analysis of plasma in COVID-19

“…We confirmed that these factors were not influencing the accuracy of our approach based on the CVs detected. CVs reported in this study confirm similar reports from other research groups ( 17 , 23 , 24 ). In this study, we were interested in determining a specific protein or a panel of proteins that could be used for COVID-19 prognosis.…”

Prognostic accuracy of MALDI mass spectrometric analysis of plasma in COVID-19

“…CVs reported in this study confirm similar reports from other research groups. 17,23,24 The overfitting issue, normally seen in these approaches, was minimized using independent datasets for the training and the validation. Moreover, the test dataset was analyzed only once based on the results from the training set avoiding subjective selection of the best results.…”

Differentiating tumor heterogeneity in formalin-fixed paraffin-embedded (FFPE) prostate adenocarcinoma tissues using principal component analysis of matrix-assisted laser desorption/ionization imaging mass spectral data

“…The technique is widely used in biomedical research to profile the distribution of proteins, peptides, and small molecules on tissue . In addition, MALDI‐IMS provides reproducible high‐resolution mass measurements of molecules in complex biological matrices such as cells and serum samples . MALDI‐IMS plays a key role in the discovery of biomarkers for the diagnosis of cancer and classification of the disease pathology .…”

“…[4][5][6] In addition, MALDI-IMS provides reproducible high-resolution mass measurements of molecules in complex biological matrices such as cells and serum samples. [7,8] MALDI-IMS plays a key role in the discovery of biomarkers for the diagnosis of cancer and classification of the disease pathology. [9,10] Given its ability to localize proteins and smaller molecules across an entire tissue section, MALDI-IMS analysis holds promise in the elucidation of molecular processes in the tumor microenvironment.…”