Reproducibility in urine peptidome profiling using MALDI-TOF

Area of Science:

• Biochemistry
• Analytical Chemistry
• Proteomics

Background:

• Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) profiling of low molecular weight peptides, or peptidome, is hindered by sample preparation and data processing variability.
• Reproducibility challenges limit the widespread clinical application of MALDI-TOF peptidome analysis.

Purpose of the Study:

• To compare centrifugal ultrafiltration and dialysis for urine peptidome pretreatment.
• To assess the effectiveness of signal limit of detection (sLOD) and data normalization in reducing MALDI-TOF variability.
• To investigate the impact of peak detection on reproducibility.

Main Methods:

• MALDI-TOF analysis was performed on urine samples pretreated with either centrifugal ultrafiltration or dialysis.
• Serial dilutions of pooled urines were analyzed to estimate sLOD.
• Six data normalization strategies (mean, median, internal standard, relative intensity, TIC, linear rescaling) were evaluated.
• A feedback signal processing approach was applied after median normalization and sLOD adjustment.

Main Results:

• Dialysis yielded superior MALDI-TOF spectra compared to ultrafiltration.
• A combined approach of median normalization and sLOD adjustment significantly reduced coefficient of variation (CV) from over 100% to 25-26%.
• This method enhanced spectral comparability and reduced data dispersion.

Conclusions:

• Dialysis is a more effective pretreatment method for urine peptidome analysis using MALDI-TOF.
• Combining sLOD adjustment with appropriate data normalization, particularly median normalization and feedback signal processing, substantially improves the reproducibility of MALDI-TOF peptidome profiling.
• These advancements enhance the reliability of MALDI-TOF for analyzing complex biological samples like urine peptidome.