Capillary electrophoresis combined with matrix-assisted laser desorption/ionization mass spectrometry; continuous sample deposition on a matrix-precoated membrane target.
Capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) were combined in an off-line arrangement to provide separation and mass analysis of peptide and protein mixtures in the attomole range. A membrane target, precoated with MALDI matrix, was used for the continuous deposition of effluent exiting from a CE device. A sample track was produced by linear movement of the target during the electrophoretic separation and this track was subsequently analyzed by MALDI/MS. The technique is effective for peptides and proteins, having limits of detection (signal-to-noise >3) of about 50 amol for neurotensin (1673 Da) and 250 amol for cytochrome c (12361 Da) and apomyoglobin (16951 Da). The electrophoretic separation achieved from the membrane target, as measured by theoretical plate numbers from the mass spectrometric data, can be as high as 80-90% of that achieved by on-line UV detection under optimal conditions, although band broadening occurs and with some loss of separation efficiency. Non-volatile buffers such as 10-50 mM phosphate can also be used in the electrophoresis process and directly deposited on the membrane. The use of post-source decay techniques is shown for peptides in the CE sample track in order to obtain sequence verification. The effectiveness of this method of integration of CE and MALDI/MS is demonstrated with both peptide and protein mixtures and with the analysis of a tryptic digest of a protein.
Zhang
H
H
Analytical Chemistry Center, Houston, Texas 77030, USA.
Caprioli
R M
RM
D001059:Apoproteins / Q000737:chemistry / Q000302:isolation & purification; D002482:Cellulose; D003574:Cytochrome c Group / Q000737:chemistry / Q000302:isolation & purification; D019075:Electrophoresis, Capillary / Q000379:methods*; D007202:Indicators and Reagents; D008567:Membranes, Artificial; D009211:Myoglobin / Q000737:chemistry / Q000302:isolation & purification; D009496:Neurotensin / Q000737:chemistry / Q000302:isolation & purification; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet
D001059:Apoproteins; D003574:Cytochrome c Group; D007202:Indicators and Reagents; D008567:Membranes, Artificial; D009211:Myoglobin; D010455:Peptides; D011506:Proteins; C010857:apomyoglobin; D009496:Neurotensin; D002482:Cellulose