6
D. R. GOODLETT et aI.
tein analysis. The signal to noise ratio and therefore working sensitivity of the mass
spectrometer is dependent on the purity of the reagents used. Organic contaminants
such as polymers extracted from plasticware by organics such as acetonitrile and
detergents left over from incompletely rinsed glassware are other common contaminants detected in the mass spectrometer. As each laboratory environment is unique,
it is difficult to suggest generic rules for optimum performance. However, a laboratory wishing to apply this technology can work out by elimination which grade of
chemicals, producer of labware and source of water are best suited.
3
Sample Introduction for Mass Spectrometry
Good sample handling techniques are critical for the success of any analytical
project. This is especially true if only a few microliters of total volume exist and
the concentration of the working sample is in the nano- to pi co molar range
(femto- to attomoles/microliter). The major problems are sample contamination
and sample loss due to adsorption to wetted surfaces. To avoid nonspecific sample loss the number of sample manipulations between digestion and mass spectrometric analysis should be kept to a minimum. For high sensitivity applications
proteolytic digests from electrophoretically separated proteins must be desalted
prior to analysis. This minimizes matrix effects from salts which compete with
peptides during the ionization process in both MALDI and ESI. For MALDI-TOF
and nano-ESI application (Shevchenko, et al. 1996; Wilm, et al. 1996) samples are
purified off-line by the use of small extraction devices such as ZIP-Tips (MilliPore). When micro-separation systems such capillary LC or CE are coupled online to ESI-MS instruments the peptide separation system serves to both, desaltl
purify and separate the analytes. For CE-MS/MS the sample is pressure injected
on a C18 cartridge (1 mm x 50 !-lm) placed at the head of the CE capillary. The
sample is desalted and then eluted with a small plug of organic solvent in to the
CE capillary for separation (Figeys et al. 1996; Figeys and Aebersold 1997). For
!-lLC we use 50-100!-lm Ld. polyimide coated capillary columns packed with C18
support to separate and desalt peptide digests (Lee et al. 1998).
To achieve high quality collision induced dissociation (CID) spectra from very
small amounts of peptides we have modified the SPE-CE-MS/MS method (Figeys
et al. 1999) by applying a variable CE voltage to decrease flow rate and thus
increase the time available for MS/MS. This peak parking method with SPE-CE
(Fig. l.2) is achieved by an automated (instrument control language) decrease in
the applied CE voltage which is initiated whenever the mass spectrometer detects
peptide ions present above a preset signal/noise ratio ( Figeys et al 1997). Simultaneously to the voltage drop the mass spectrometer is switched from scanning to
CID mode. The concentration dependent nature of ESI (Goodlett, et al. 1993)
allows one to fragment peptides of low abundance by reducing the flow of liquid
without sacrificing sensitivity. Peak parking is advantageous because ion selection by signal/noise works well for abundant peptides in a digest but fails, due to
a lack of time, to select lower abundance peptides co-eluting with abundant peptides. Therefore, if complex peptide mixtures are analyzed the lower abundance
species, frequently the most interesting biologically, go undetected even though
D. R. GOODLETT et aI.
tein analysis. The signal to noise ratio and therefore working sensitivity of the mass
spectrometer is dependent on the purity of the reagents used. Organic contaminants
such as polymers extracted from plasticware by organics such as acetonitrile and
detergents left over from incompletely rinsed glassware are other common contaminants detected in the mass spectrometer. As each laboratory environment is unique,
it is difficult to suggest generic rules for optimum performance. However, a laboratory wishing to apply this technology can work out by elimination which grade of
chemicals, producer of labware and source of water are best suited.
3
Sample Introduction for Mass Spectrometry
Good sample handling techniques are critical for the success of any analytical
project. This is especially true if only a few microliters of total volume exist and
the concentration of the working sample is in the nano- to pi co molar range
(femto- to attomoles/microliter). The major problems are sample contamination
and sample loss due to adsorption to wetted surfaces. To avoid nonspecific sample loss the number of sample manipulations between digestion and mass spectrometric analysis should be kept to a minimum. For high sensitivity applications
proteolytic digests from electrophoretically separated proteins must be desalted
prior to analysis. This minimizes matrix effects from salts which compete with
peptides during the ionization process in both MALDI and ESI. For MALDI-TOF
and nano-ESI application (Shevchenko, et al. 1996; Wilm, et al. 1996) samples are
purified off-line by the use of small extraction devices such as ZIP-Tips (MilliPore). When micro-separation systems such capillary LC or CE are coupled online to ESI-MS instruments the peptide separation system serves to both, desaltl
purify and separate the analytes. For CE-MS/MS the sample is pressure injected
on a C18 cartridge (1 mm x 50 !-lm) placed at the head of the CE capillary. The
sample is desalted and then eluted with a small plug of organic solvent in to the
CE capillary for separation (Figeys et al. 1996; Figeys and Aebersold 1997). For
!-lLC we use 50-100!-lm Ld. polyimide coated capillary columns packed with C18
support to separate and desalt peptide digests (Lee et al. 1998).
To achieve high quality collision induced dissociation (CID) spectra from very
small amounts of peptides we have modified the SPE-CE-MS/MS method (Figeys
et al. 1999) by applying a variable CE voltage to decrease flow rate and thus
increase the time available for MS/MS. This peak parking method with SPE-CE
(Fig. l.2) is achieved by an automated (instrument control language) decrease in
the applied CE voltage which is initiated whenever the mass spectrometer detects
peptide ions present above a preset signal/noise ratio ( Figeys et al 1997). Simultaneously to the voltage drop the mass spectrometer is switched from scanning to
CID mode. The concentration dependent nature of ESI (Goodlett, et al. 1993)
allows one to fragment peptides of low abundance by reducing the flow of liquid
without sacrificing sensitivity. Peak parking is advantageous because ion selection by signal/noise works well for abundant peptides in a digest but fails, due to
a lack of time, to select lower abundance peptides co-eluting with abundant peptides. Therefore, if complex peptide mixtures are analyzed the lower abundance
species, frequently the most interesting biologically, go undetected even though