High-sensitivity peptide mapping by micro-LC with on-line membrane blotting and subsequent detection by scanning-IR-MALDI mass spectrometry.
A novel approach to the on-line mass determination of peptides from digested proteins by scanning infrared matrix-assisted laser desorption/ionization (scanning-IR-MALDI) is described. The peptides were continuously collected directly onto a PVDF (polyvinylidene fluoride) strip during a HPLC run. Individual peptides were detected by lining up the PVDF strip with the UV trace from the HPLC run, using visible dye markers as reference points. The local resolution of the peptides on the PVDF membrane is preserved during matrix incubation for MALDI-MS as shown by comparing the UV chromatogram and the total ion current (TIC) from an on-line coupled electrospray ionization (ESI) mass spectrometer with the scanning-IR-MALDI data from the corresponding areas on the PVDF strip. The intensities of the mass profiles obtained by scanning-IR-MALDI reflect the amount of peptides present on the PVDF strip. The higher sensitivity of IR-MALDI-MS yielded mass information not detectable by ESI-MS. After the scanning-IR-MALDI experiment, the same membrane strip can be used directly for automated Edman degradation. Comparable initial and repetitive yields were obtained for blotted peptides with and without matrix incubation.
Eckerskorn
C
C
Max-Planck-Institute for Biochemistry, Martinsried, Germany.
Strupat
K
K
Kellermann
J
J
Lottspeich
F
F
Hillenkamp
F
F
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D008567:Membranes, Artificial; D008832:Microchemistry; D010446:Peptide Fragments / Q000032:analysis*; D010449:Peptide Mapping / Q000379:methods*; D011145:Polyvinyls; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013055:Spectrophotometry, Infrared / Q000379:methods; D013056:Spectrophotometry, Ultraviolet; D005465:Fluorocarbon Polymers
D008567:Membranes, Artificial; D010446:Peptide Fragments; D011145:Polyvinyls; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers