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.

ABSTRACT

To minimize low-quantity sample handling for protein sequencing and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, a system consisting of an HPLC interfaced to an automated blotting device was used for off-line sample collection. Typically, protein digests are separated by reverse-phase HPLC and the resulting peptide fractions are pooled, concentrated, and then subjected to N-terminal sequence analysis. Obtaining unambiguous sequence from peptides derived from protein digestion at subpicomole levels requires careful sample handling to prevent loss of sample. In cases where multiple sequences are present, a secondary method such as mass spectrometry is needed to confirm the identity of the peptides. To minimize sample handling, commercial 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 mass spectrometry, we investigated the use of MALDI-MS compatible membranes such as Teflon and polyethylene (PE) as the blotting media for fraction collection. Using a panel of standard peptides as well as protein digests, we demonstrate that peptides separated by capillary HPLC can be collected directly onto Teflon or PE and detected into the femtomole range. Furthermore, detailed sequence analysis could be obtained by postsource decay fragmentation spectra of individual peptides blotted onto either PE or Teflon. Due to the high sensitivity of the MALDI-MS from these membranes, it was discovered that the small amount of peptide that passed through the PVDF membrane during a collection of peptides for N-terminal sequencing was sufficient to be collected and mass analyzed from a second underlying MALDI-MS compatible membrane. Therefore, from a single HPLC separation, samples could be collected onto both PVDF for traditional N-terminal sequencing and PE or Teflon for MALDI-MS. We demonstrate the general utility of this method for sequencing peptides from a tryptic digestion at subpicomole levels and for identifying unknown proteins separated by 2-dimensional gel electrophoresis. 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.