HIGH-SENSITIVITY PEPTIDE-MAPPING BY MICRO-LC WITH ONLINE MEMBRANE BLOTTING AND SUBSEQUENT DETECTION BY SCANNING-IR-MALDI MASS-SPECTROMETRY

A novel approach to the on-line mass determination of peptides from di gested proteins by scanning infrared matrix-assisted laser desorption/ ionization (scanning-IR-MALDI) is described. The peptides were continu ously collected directly onto a PVDF (polyvinylidene fluoride) strip d uring a HPLC run. Individual peptides were detected by lining up the P VDF strip with the UV trace from the HPLC run, using visible dye marke rs as reference points. The local resolution of the peptides on the PV DF membrane is preserved during matrix incubation for MALDI-MS as show n by comparing the UV chromatogram and the total ion current (TIC) fro m an on-line coupled electrospray ionization (ESI) mass spectrometer w ith the scanning-IR-MALDI data from the corresponding areas on the PVD F strip. The intensities of the mass profiles obtained by scanning-IR- MALDI reflect the amount of peptides present on the PVDF strip. The hi gher sensitivity of IR-MALDI-MS yielded mass information not detectabl e by ESI-MS. After the scanning-IR-MALDI experiment, the same membrane strip can be used directly for automated Edman degradation. Comparabl e initial and repetitive yields were obtained for blotted peptides wit h and without matrix incubation.