Homogeneous sample preparation for automated high throughput analysis withmatrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

This work presents a simple method for obtaining homogeneous sample surface s in matrix-assisted laser desorption/ionisation time-of-flight mass spectr ometry (MALDI-TOFMS) for the automated analysis of peptides and proteins. T he sample preparation method is based on applying the sample/matrix mixture onto a pre-deposited highly diluted matrix spot. The pre-deposited crystal s act as seeds for the new sample containing crystals which become much sma ller in size and more evenly distributed than with conventional methods. Th is 'seed-layer' method was developed, optimised and compared with the dried -droplet method using peptides and proteins in the 1000-20000 Da range. The seed-layer method increases the surface homogeneity, spot to spot reproduc ibility and sample washability as compared with the commonly used dried-dro plet method. This methodology is applicable to alpha-cyanohydroxycinnamic a cid, sinapinic acid and ferulic acid, which all form homogeneous crystal su rfaces. Within-spot variation and between-spot variation was investigated u sing statistics at a 95% confidence level (n = 36). The statistical values were generated from more than 5000 data points collected from 500 spectra, More than 90% of the sample locations results in high intensity spectra wit h relatively low standard deviations (RSDs), Typically obtained data showed an RSD of 19-35% within a sample spot as well. as in-between spots for pro teins, and an RSD of less than or equal to 50% for peptides, Linear calibra tion curves were obtained within one order of magnitude using internal cali bration with a point-RSD of 3% (n = 10), The sample homogeneity allows mass spectra (average of 16 laser shots) to be obtained on each individual samp le within 15 sec, whereby a 100 spot target plate can be run in 25 min. Hig h density target plates using the seed-layer method were prepared by spotti ng approximate to 100 picoliter droplets onto the target, resulting in samp le spots less than or equal to 500 mu m in diameter using a flow-through pi ezo-electric micro-dispenser. By using this automated sample preparation st ep lower standard deviations are obtained in comparison to manually prepare d samples. Copyright (C) 1999 John Wiley & Sons, Ltd.