Applications of in-source fragmentation of protein ions for direct sequence analysis by delayed extraction MALDI-TOF mass spectrometry.
In matrix-assisted laser desorption/ionization of proteins, there exists a certain amount of fast metastable decay immediately after laser irradiation. The fragment ions thus formed can be resolved and their m/z values measured accurately by employing delayed extraction linear time-of-flight mass spectrometry. At higher than threshold laser fluences, proteins exhibit a series of fragment ions providing useful sequence information. We also observe that when moderate amounts of salts are present in the sample with sinapinic acid being the matrix, the intensities of cn ions (N-terminal fragments) are enhanced compared to other types of fragment ions. This enhancement in cn ion signals allows direct sequencing of proteins. The cn ions are completely absent when Xxx-Pro bonds are encountered and are of lower intensity when Xxx-Gly bonds are involved. Further, the cn ion series is interrupted at Xxx-Cys, when the cysteine is involved in a disulfide bond. Upon reduction of the disulfide bonds, the series continues and information is available for longer stretches. Using 10-20 pmol of recombinant proteins, sometimes contiguous sequence information up to 70 residues is obtained in a matter of minutes. Applications of the technique to some recombinant proteins with intra- or interchain disulfide linkages are presented.
Katta
V
V
Amgen Inc., Thousand Oaks, California 91320, USA. vkatta@amgen.com
Chow
D T
DT
Rohde
M F
MF
D003373:Coumaric Acids; D004220:Disulfides / Q000737:chemistry; D005998:Glycine / Q000737:chemistry; D007477:Ions; D020738:Leptin; D008970:Molecular Weight; D011392:Proline / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D003373:Coumaric Acids; D004220:Disulfides; D007477:Ions; D020738:Leptin; D011506:Proteins; C073734:sinapinic acid; D011392:Proline; D005998:Glycine