Signal amplification using "spot on-a-chip" technology for the identification of proteins via MALDI-TOF MS

The presented "spot-on-a-chip" technology enables easy enrichment of sample s in the low nanomolar (1-5 nM) range and provides a fast and reliable auto mated sample preparation method for performing matrix-assisted laser desorp tion/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis wi th high sensitivity and throughput. Through microdispensing, which allows a ccurate deposition of 60-pL droplets, dilute samples were enriched by makin g multiple droplet depositions in nanovials. The sample was confined to a d efined spot area (300 x 300 mum), and multiple depositions increase the sur face density of analyte in the nanovial, thereby providing detection of low attomole levels. The impact of the nanovial geometry with respect to the M ALDI-TOF MS resolution for peptides deposited in the microfabricated silico n vials was investigated and the optimal geometry and size were determined. The spot-on-a-chip technology, that is, the combination of microdispensing , micromachined silicon nanovials and on-spot enrichment provides a signal amplification of at least 10-50 times as compared to an ordinary sample pre paration. The linearity of the enrichment effect is shown by the analysis o f a peptide mixture at the 5 nM level. The signal amplification provided by the spot-on-a-chip enrichment is demonstrated by the analysis of relevant biological samples, interleukin-8 from a spiked cell supernatant, and by su ccessful protein identification of an excised spot from a high-sensitivity silver-stained two-dimensional electrophoresis gel separation.