Laboratory methods to improve SELDI peak detection and quantitation

Abstract

Background: Protein profiling with surface-enhanced laser desorption-ionisation time-of-flight mass spectrometry (SELDI-TOF MS) is a promising approach for biomarker discovery. Some candidate biomarkers have been identified using SELDI-TOF, but validation of these can be challenging because of technical parameters that effect reproducibility. Here we describe steps to improve the reproducibility of peak detection.

Methods: SELDI-TOF mass spectrometry was performed using a system manufactured by Ciphergen Biosystems along with their ProteinChip System. Serum from 10 donors was pooled and used for all experiments. Serum was fractionated with Expression Difference Mapping kit-Serum Fractionation from the same company and applied to three different ProteinChips. The fractionations were run over a one month period to examine the contribution of sample batch and time to peak detection variability. Spectra were processed and peaks detected using the Ciphergen Express software and variance measured.

Results: Experimental parameters specific to the serum fraction and ProteinChip, including spot protocols (laser intensity and detector sensitivity) were optimized to decrease peak detection variance. Optimal instrument settings, regular calibration along with controlled sample handling and processing nearly doubled the number of peaks detected and decreased intensity variance.

Conclusion: This report assesses the variation across fractionated sera processed over a one-month period. The optimizations reported decreased the variance and increased the number of peaks detected.

Figure 1

Outline of the experimental workflow for SELDI-TOF optimization. QC serum was fractionated and run as 6 replicates of fractions F1, F3, F4, and F6 on IMAC, CM10 LS and H50 ProteinChips; as 6 replicates of F3 and F4 on CM10 HS and 6 replicates of F5 on CM10 LS ProteinChips.