Guanidino labeling derivatization strategy for global characterization of peptide mixtures by liquid chromatography matrix-assisted laser desorption/ionization mass spectrometry.
Guanidination performed with isotopic isoforms of O-methylisourea was used in combination with reversed-phase liquid chromatography (LC) matrix-assisted laser desorption/ionization to characterize, both qualitatively and quantitatively, protein mixtures. Synthesis of (13)C- and (15)N(2)-labeled O-methylisourea sulfate produces a molecule that is 3 Da heavier than the light isotopic variant. Protein mixtures containing identical components in different concentration are pooled together following parallel derivatization. Relative quantification of protein mixtures is achieved by mass spectrometry. A difference of 3 Da allows negligible interference between the two isotopic clusters for quantification of peptides up to 1400 Da. Under these conditions, the chromatographic resolution achieved allows separation of different pairs of derivatized peptides without altering the retention time of structurally identical isotopic isoforms. Concomitant isolation of both chemically modified precursors is followed by tandem mass analysis. Activation of the ions via collisions with an inert gas produces isotopically derivatized fragment ions, which appear as doublets in the product ion spectrum. Since the modification occurs on the C-terminal lysine, ions incorporating the guanidino moiety on the C-terminus can be distinguished from those containing the original unmodified peptide N-terminus. Knowledge of the location of the proton can be beneficial to data interpretation and peptide sequencing.
Brancia
Francesco L
FL
Shimadzu Research Laboratory (Europe), Wharfside, Trafford Wharf Road, Manchester, M17 1GP, UK. Francesco.Brancia@srlab.co.uk
Montgomery
Helen
H
Tanaka
Koichi
K
Kumashiro
Sumio
S
D002853:Chromatography, Liquid / Q000379:methods*; D006146:Guanidines / Q000737:chemistry*; D007553:Isotope Labeling; D008239:Lysine / Q000737:chemistry; D008782:Methylurea Compounds / Q000737:chemistry; D010455:Peptides / Q000032:analysis*; D020033:Protein Isoforms / Q000737:chemistry; D011506:Proteins / Q000032:analysis*; D017421:Sequence Analysis / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013431:Sulfates / Q000737:chemistry
D006146:Guanidines; D008782:Methylurea Compounds; D010455:Peptides; D020033:Protein Isoforms; D011506:Proteins; D013431:Sulfates; C002249:O-methylisourea; D008239:Lysine