Increased sensitivity of tryptic peptide detection by MALDI-TOF mass spectrometry is achieved by conversion of lysine to homoarginine.
Mass spectrometric techniques for identification of proteins by "mass fingerprinting" (matching the masses of tryptic peptides from a protein digest to the theoretical peptides in a database) such as matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) are rapidly growing in popularity as the demand for high throughput analysis of the proteome increases. This is due, in part, to the ability to automate the technique and the rapid rate with which mass spectra may be acquired. An important factor in the accuracy of the technique is the number of tryptic peptides that are identified in the various searching algorithms that exist. The greater sequence coverage of the parent protein that is obtained, the higher the level of confidence in the identification that is determined. One impediment to high levels of sequence coverage is the bias of MALDI-TOF mass spectrometry to arginine-containing peptides. Increasing the sensitivity to lysine-containing peptides should increase the sequence coverage obtained. In order to achieve this result we have developed conditions to modify the epsilon-amine group of lysine in tryptic peptides with O-methylisourea. The conditions utilized result in the conversion of lysine to homoarginine with no modification of the amine terminus of the peptides. The sensitivity of MALDI-TOF mass spectrometry detection of peptides was increased dramatically following modification. The modification chemistry may be applied to tryptic peptide mixtures prior to desalting and spotting onto MALDI-TOF plates. This technique will be particularly useful for identifying proteins with a high lysine/arginine ratio.
Hale
J E
JE
Research Technologies and Proteins Division, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana, 46285, USA. Hale_John_E@Lilly.com
Butler
J P
JP
Knierman
M D
MD
Becker
G W
GW
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000032:analysis / Q000737:chemistry; D002417:Cattle; D003574:Cytochrome c Group / Q000032:analysis / Q000737:chemistry; D045304:Cytochromes c; D006709:Homoarginine / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry*; D008782:Methylurea Compounds / Q000494:pharmacology; D010449:Peptide Mapping / Q000379:methods*; D010455:Peptides / Q000032:analysis*; D017421:Sequence Analysis; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D001059:Apoproteins; D003574:Cytochrome c Group; D008782:Methylurea Compounds; D010455:Peptides; D006709:Homoarginine; C002249:O-methylisourea; D012710:Serum Albumin, Bovine; D045304:Cytochromes c; D008239:Lysine