title: Guanidination of tryptic peptides without desalting for matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis.
abstract: Derivatizations that enhance mass spectral quality often require desalting, which presents as a bottleneck in matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-proteomics. Guanidination, which converts lysine to homoarginine, an arginine analogue, can increase detection of those peptides 5-15-fold. Our aim was to improve guanidination by using a novel reagent, O-methylisourea-freebase. In a simple reaction, interfering salts were removed prior to guanidination. Freebase preparation took about 30 min and could be applied to samples all at once as opposed to desalting samples one-by-one for 5 min each. For freebase guanidinated BSA tryptic peptides, more than 6-times the peptides were observed relative to tryptic peptides or those guanidinated with the conventional reagent, O-methylisourea hemisulfate. Peptide signals increased more than 10-fold relative to those from guanidination with the conventional reagent and were equivalent to those from conventional guanidination with desalting. In addition, freebase guanidination allowed for a lower limit of detection when combined with another derivatization, N-terminal sulfonation, as evidenced by tandem mass spectrometry (MS/MS) fragmentation analysis of in-gel digests of cytochrome c. Freebase guanidination of rat lung proteins after 2-D gel electrophoresis allowed for identification of all tested protein spots regardless of protein characteristics (MW or pI) or abundance. Co-derivatization with N-terminal sulfonation confirmed the identity of low-abundance proteins in 2-D gel spots that contained more than one protein. The freebase guanidination reagent is simple to prepare and to implement. Desalting is not needed prior to MALDI-TOF MS. Freebase guanidination effectively increases the dynamic range of detection of lysine-containing peptides while decreasing the work needed for sample preparation.
authors: Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa , 1955 East-West Road, Honolulu, Hawaii 96822, United States.
authors: Margaret R
authors: MR
authors: Baker
authors: Qing X
authors: QX
authors: Li
mesh_terms: D000818:Animals; D002417:Cattle; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D019791:Guanidine / Q000032:analysis* / Q000378:metabolism; D010455:Peptides / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D010455:Peptides; D012710:Serum Albumin, Bovine; D019791:Guanidine
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