Applications of In-Source Fragmentation of Protein Ions for Direct Sequence Analysis by Delayed Extraction MALDI-TOF Mass Spectrometry

Abstract

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.

Flexible Xxx^|^#8211;Asp/Asn and Gly^|^#8211;Xxx Residues of Equine Cytochrome c in Matrix-Assisted Laser Desorption/Ionization In-Source Decay Mass Spectrometry

“…Early MALDI-ISD studies of backbone exibility of protein showed that the N-C α bond between Gly35 and His36 residues lying in a turn region between α-helix segments 21-35 and 37-40 of myoglobins was more susceptible to ISD than the rest of the protein, 26) while the N-C α bond between Xxx-Gly, Xxx-Val and Xxx-Ile residues was less susceptible to ISD than between other amino acid residues. 27,28) We have recently reported that in ISD spectra of equine myoglobin, equine cytochrome c and bovine serum albumin the N-C α bonds between Xxx-Asp, Xxx-Asn and Gly-Xxx residues, which are preferred in exible secondary structures such as turn and bend than in intra-molecular hydrogen-bonded structures such as α-helix, are more susceptible to MALDI-ISD than other residues. 29) It is therefore of interest to examine the relationships between the amino acids susceptible to MALDI-ISD and the rate of HDX of the backbone amides of protein, because amino acid residues free from intra-molecular hydrogen-bonded helix and sheet would be expected to have enhanced hydrogen radical attachment and HDX.…”

MALDI In-Source Decay of Protein

“…61,62) erefore, MALDI-ISD can be used for the topdown sequencing of unknown and post-translational modied proteins. 8,[21][22][23][24][25][26][27][28][29][30]61,62) It should be stressed that the model for MALDI-ISD was constructed on the basis of X-ray crystallography ndings and SPM analyses of matrix crystals, as well as the use of isotope-labelled peptides.…”

“…19) ey reported the observation of unusual fragment ions c and z+2 assigned by Biemann's nomenclature. 20) Subsequently, several groups reported on the MALDI-ISD data of peptides and proteins from the standpoints of practical, 8,[21][22][23][24][25][26][27][28][29][30] basic [7][8][9][10][31][32][33] and review interests, 34) as summarized in Table 1. Recent advances in the fragmentation and theoretical considerations of MALDI-ISD have been reviewed by Asakawa 35) and Moon et al 36) We also found fragment ions c and z+2 re ecting the amino acid sequence in a MALDI mass spectrum of angiotensin I independent of Brown and Lennon's report, 6) in the course of experiments directed at the in uence of laser uence on the appearance and peak resolution of an analyte signal [A+H] + in the late 1997s.…”

Top-down, bottom-up, and side-to-side proteomics with virtual 2-D gels

“…On MALDI-equipped platforms, in-source dissociation provides limited access to topdown sequencing capability [5,11,14,15,[37][38][39][40][41][42]. ISD has been demonstrated with ions desorbed directly from an IPG strip onto which Escherichia coli whole cell lysates had been focused [5].…”