CHAPTER 8
Elucidation of Functionally Significant Structural
Modifications by Matrix-Assisted Laser Desorption/
Ionization Time-of-Flight Mass Spectrometry
with Post-Source Decay Analysis
J. J. GORMAN!, B. L. FERGUSON!, S. LOPATICKI!, J. J. PITT!,
A. W. PURCELL 2 and C. J. MORROW 3
1
Introduction
Analysis of peptides and proteins by mass spectrometry (MS) became accessible
to non-specialist mass spectroscopists with the advent of soft ionization techniques such as plasma desorption (Macfarlane and Thorgerson 1976) and fastatom bombardment (FAB) (Barber et al. 1981). These techniques enabled ionization and detection of pseudomolecular ions of non-derivatized peptides and
small proteins using modest quantities of analytes (Barber et al. 1982; Sundqvist
et al. 1984). Sequence data were occasionally evident in FAB ionization spectra as
a result of in-source fragmentation (Barber et al. 1982) or post-source collisional
activation (Desiderio and Katakuse 1983; Katakuse and Desiderio 1983). Generation of extensive fragmentation was usually achieved using multi analyser instruments with a collision cell between an analyser used to select parent ions for fragmentation and a subsequent analyser used to define daughter ions (Biemann
1990; Carr et al. 1990; Hunt et al. 1990). Consequently, high performance tandem
double focussing sector and triple quadrupole instruments with extended performance characteristics were developed.
Development of Matrix-Assisted Laser Desorption/Ionization (MALDI) (Karas
and Hillenkamp 1988) and Electrospray Ionization (ESI) (Fenn et al. 1989) led to
more general utilization of mass spectrometry by protein chemists. This was
largely due to greater sensitivities and extended mass range capabilities of these
ionization techniques. However, improved user friendliness of data systems and
mass analysers, mainly Time-Of-Flight (TOF) and quadrupoles, has also promoted more universal use. ESI in conjunction with triple quadrupole analysers
has produced sequence data on extremely small quantities of peptides (Wilm et
al. 1996) as well as molecular weight information on peptides and small proteins
(Fenn et al. 1989). Use of Fourier transform ion-cyclotron resonance, ion-trap
and hybrid quadrupole TOF or Q-TOF instruments has further enhanced the
scope for producing peptide sequence data in conjunction with ESI.
Use of reflecting TOF instruments has revealed that MALDI generated peptide
ions undergo fragmentation by post-source decay (PSD) during their relatively
1 Biomolecular Research Institute and
2 University of Melbourne, 343 Royal Parade, Parkville, Vic. 3052, Australia and
3 Ross Breeders, Scotland, UK.
Elucidation of Functionally Significant Structural
Modifications by Matrix-Assisted Laser Desorption/
Ionization Time-of-Flight Mass Spectrometry
with Post-Source Decay Analysis
J. J. GORMAN!, B. L. FERGUSON!, S. LOPATICKI!, J. J. PITT!,
A. W. PURCELL 2 and C. J. MORROW 3
1
Introduction
Analysis of peptides and proteins by mass spectrometry (MS) became accessible
to non-specialist mass spectroscopists with the advent of soft ionization techniques such as plasma desorption (Macfarlane and Thorgerson 1976) and fastatom bombardment (FAB) (Barber et al. 1981). These techniques enabled ionization and detection of pseudomolecular ions of non-derivatized peptides and
small proteins using modest quantities of analytes (Barber et al. 1982; Sundqvist
et al. 1984). Sequence data were occasionally evident in FAB ionization spectra as
a result of in-source fragmentation (Barber et al. 1982) or post-source collisional
activation (Desiderio and Katakuse 1983; Katakuse and Desiderio 1983). Generation of extensive fragmentation was usually achieved using multi analyser instruments with a collision cell between an analyser used to select parent ions for fragmentation and a subsequent analyser used to define daughter ions (Biemann
1990; Carr et al. 1990; Hunt et al. 1990). Consequently, high performance tandem
double focussing sector and triple quadrupole instruments with extended performance characteristics were developed.
Development of Matrix-Assisted Laser Desorption/Ionization (MALDI) (Karas
and Hillenkamp 1988) and Electrospray Ionization (ESI) (Fenn et al. 1989) led to
more general utilization of mass spectrometry by protein chemists. This was
largely due to greater sensitivities and extended mass range capabilities of these
ionization techniques. However, improved user friendliness of data systems and
mass analysers, mainly Time-Of-Flight (TOF) and quadrupoles, has also promoted more universal use. ESI in conjunction with triple quadrupole analysers
has produced sequence data on extremely small quantities of peptides (Wilm et
al. 1996) as well as molecular weight information on peptides and small proteins
(Fenn et al. 1989). Use of Fourier transform ion-cyclotron resonance, ion-trap
and hybrid quadrupole TOF or Q-TOF instruments has further enhanced the
scope for producing peptide sequence data in conjunction with ESI.
Use of reflecting TOF instruments has revealed that MALDI generated peptide
ions undergo fragmentation by post-source decay (PSD) during their relatively
1 Biomolecular Research Institute and
2 University of Melbourne, 343 Royal Parade, Parkville, Vic. 3052, Australia and
3 Ross Breeders, Scotland, UK.