COUPLING CAPILLARY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY TO MATRIX-ASSISTED-LASER-DESORPTION IONIZATION MASS-SPECTROMETRY AND N-TERMINAL SEQUENCING OF PEPTIDES VIA AUTOMATED MICROBLOTTING ONTO MEMBRANE SUBSTRATES/

To minimize low-quantity sample handling for protein sequencing and ma trix-assisted laser desorption/ ionization (MALDI) mass spectrometry, a system consisting of an HPLC interfaced to an automated blotting dev ice was used for off-line sample collection. Typically, protein digest s are separated by reverse-phase HPLC and the resulting peptide fracti ons are pooled, concentrated, and then subjected to N-terminal sequenc e analysis. Obtaining unambiguous sequence from peptides derived from protein digestion at subpicomole levels requires careful sample handli ng to prevent loss of sample. In cases where multiple sequences are pr esent, a secondary method such as mass spectrometry is needed to confi rm the identity of the peptides. To minimize sample handling, commerci al microblotting instruments have become available to deposit peptides directly onto polyvinylidene difluoride (PVDF) membrane for automated N-terminal sequence analysis. In order to adapt this technology to ma ss spectrometry, we investigated the use of MALDI-MS compatible membra nes such as Teflon and polyethylene (PE) as the blotting media for fra ction collection. Using a panel of standard peptides as well as protei n digests, we demonstrate that peptides separated by capillary HPLC ca n be collected directly onto Teflon or PE and detected into the femtom ole range. Furthermore, detailed sequence analysis could be obtained b y postsource decay fragmentation spectra of individual peptides blotte d onto either PE or Teflon. Due to the high sensitivity of the MALDI-M S from these membranes, it was discovered that the small amount of pep tide that passed through the PVDF membrane during a collection of pept ides for N-terminal sequencing was sufficient to be collected and mass analyzed from a second underlying MALDI-MS compatible membrane. There fore, from a single HPLC separation, samples could be collected onto b oth PVDF for traditional N-terminal sequencing and PE or Teflon for MA LDI-MS. We demonstrate the general utility of this method for sequenci ng peptides from a tryptic digestion at subpicomole levels and for ide ntifying unknown proteins separated by S-dimensional gel electrophores is. The ability to generate both N-terminal sequence and confirmatory mass information from multiple peptides in a single separation greatly improves the reliability and the accuracy of protein characterization at subpicomole levels. (C) 1998 Academic Press.