Parallel sample preparation of proteins, from crude samples to crystals ready for MALDI-MS, in an integrated microfluidic system.
A microfluidic structure is presented where selective capture of proteins in complex samples, followed by clean-up, enzymatic processing, and MALDI-MS sample preparation of peptides generated, can be performed. The structure uses an affinity column to capture the protein while all other components in the sample are disposed of. The protein of interest is then eluted from the affinity column and captured on a second column on which the enzymatic processing is performed. Salts and hydrophilic contaminants are then removed before the products from the enzymatic reaction are eluted together with a suitable MALDI matrix and the solvent evaporated in a designated MALDI target structure. All steps can be performed automatically in 54 parallel microstructures on a microfluidic compact disc. The process is demonstrated by the selective capture and tryptic digest of recombinant IgG molecules from samples containing other proteins: an excess of bovine serum albumin or spent cell culture media.
Tran
Thi Thuy
TT
Department of Analytical Chemistry, Stockholm University, SE-106 91 Stockholm, Sweden.
Inganäs
Mats
M
Ekstrand
Gunnar
G
Thorsén
Gunnar
G
D000818:Animals; D002417:Cattle; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification / Q000378:metabolism; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods*; D010449:Peptide Mapping / Q000379:methods*; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D007074:Immunoglobulin G; D011994:Recombinant Proteins; D014357:Trypsin