Protein identification platform utilizing micro dispensing technology interfaced to matrix-assisted laser desorption ionization time-of-flight mass spectrometry

An integrated protein microcharacterization/identification platform has bee n developed. The system has been designed to show a high flexibility in ord er to tackle challenging analytical problems. The platform comprises a cool ed microautosampler, an integrated system for microcolumn HPLC, and a capil lary reversed-phase column that is interfaced to matrix-assisted laser deso rption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) system vi a a low internal volume flow-through microdispenser. The chromatographic se paration is continuously transferred onto a MALDI target plate as discrete spots as the dispenser ejects bursts of droplets of the column effluent in a precise array pattern. A refrigerated microfraction collector was coupled to the outlet of the flow-through microdispenser enabling enrichment and r e-analysis of interesting fractions. The use of target plates pre-coated wi th matrix simplified and increased the robustness of the system. By includi ng a separation step prior to the MALDI-TOF-MS analysis and hereby minimizi ng suppression effects allowed us to obtain higher sequence coverage of pro teins compared to conventional MALDI sample preparation methodology. Additi onally, synthetic peptides corresponding to autophosphorylated forms of the tryptic fragment 485-496 (ALGADDSTYTAR) of tyrosine kinase ZAP-70 were ide ntified at sensitivities reaching 150 amol. (C) 2000 Elsevier Science B.V. All rights reserved.