Digestion protocol for small protein amounts for nano-HPLC-MS(MS) analysis.

A miniaturized tryptic digestion protocol for protein analysis has been developed, which works well for small amounts of proteins using small volume of reagents. The protocol starts from 10μL sample volume with total protein content in the low pmol or fmol range (alternatively expressed, in the low ng range). After adding various reagents the total volume of the tryptic digest will increase to 15μL only. This is especially advantageous for nano-HPLC-MS or MALDI analysis which requires (and allows) analysis of few μL aliquots only. Efficiency of the protocol was tested using nano-HLPC-MS(MS). The results show that the developed miniaturized digestion protocol performs at least as well, possibly even better, than conventional protocols using large sample amounts; and is far superior to digestion performed in larger volumes followed by solvent evaporation/resolvation. This is reflected both in signal intensities in MS and in the number of proteins identified by MS/MS.