Future Directions

The analysis of biologically derived peptides and proteins will continue to be dominated by the need for rapid analyses of complex mixtures at increasingly lower levels of analytes. This is particularly true for analytes in arenas such as clinical chemistry, toxicology, and forensics. Furthermore, developing practices in the area of proteomics will inevitably be more demanding of the sensitivity of analytical methods. Current practices demand that the sensitivity of protein sequencing methods is comparable to that of slab gel silver-staining techniques. While these approaches are yielding results, it is likely that many biologically significant proteins will not be detected or characterized. However, at the sensitivity levels that will be required in this area of research, sample manipulations become a major challenge. Both analyte losses and sample contamination by ubiquitous human proteins (such as keratin) become significant issues at picomolar concentrations. Automated preparation and analysis of low volume, low concentration samples of high complexity will be required to overcome these issues. Clean-room technology is also likely to become a major requirement of these investigations. Figeys and Abersold (17) recently proposed a solution to these issues. These investigators have suggested the use of coupled microfabricated, integrated analytical modules with mass spectrometry for this application, and they have recently proposed MFD-MS as the acronym for this technology. While research of these devices is in its infancy, such miniaturized approaches coupled with a sensitive mass spectrometer could

Fig. 8. Identification of human renal dialysate protein obtained from 200 L of dialysate by mPC-CE-MS and tandem MS. (A) mPC-CE-MS analysis of active fraction shown to inhibit phosphate uptake in renal epithelial cells. 20 ^L of active fraction pressure injected onto an SDB membrane. Protein was eluted with 80:20 MeOH:H2O. Separation was carried out on a bare fused silica capillary (50 ^m x 78 cm) at a voltage of 15 kV. MS was performed on MAT 900 under similar conditions to those described in Fig. 7. (B) mPC-CE-MS/MS of precursor ion MH22+ = 574.5 derived from lysine-C proteolytic digestion of protein (Mr= 11,729 Daltons). 60 ^L of lysine-C digest of protein was loaded onto an SDB membrane. Peptides were eluted with 60 nL 80:20 MeOH:H2O between a LSB of 60 nL of 1% NH4OH and TSB of 90 nL 2 mM NH4OAc. All other conditions of CE capillary and MS/MS as described in Figs. 5 and 6, respectively.

Fig. 8. Identification of human renal dialysate protein obtained from 200 L of dialysate by mPC-CE-MS and tandem MS. (A) mPC-CE-MS analysis of active fraction shown to inhibit phosphate uptake in renal epithelial cells. 20 ^L of active fraction pressure injected onto an SDB membrane. Protein was eluted with 80:20 MeOH:H2O. Separation was carried out on a bare fused silica capillary (50 ^m x 78 cm) at a voltage of 15 kV. MS was performed on MAT 900 under similar conditions to those described in Fig. 7. (B) mPC-CE-MS/MS of precursor ion MH22+ = 574.5 derived from lysine-C proteolytic digestion of protein (Mr= 11,729 Daltons). 60 ^L of lysine-C digest of protein was loaded onto an SDB membrane. Peptides were eluted with 60 nL 80:20 MeOH:H2O between a LSB of 60 nL of 1% NH4OH and TSB of 90 nL 2 mM NH4OAc. All other conditions of CE capillary and MS/MS as described in Figs. 5 and 6, respectively.

provide the detection limits required in the next generation of proteomic studies. Furthermore, with careful design of the array, on-chip protein isolation, digestion, and analysis could be accommodated in this nano-environ-ment. However, the use of nano-technologies will provide new challenges. First, creative coupling of the macro world of biochemistry to the nano-analytical environment will be required. In this regard, the lessons learned from the development of mPC-CE-MS and SPC-CE technologies should aid these studies. Particularly in the area of reducing potentially long sample injection times, caused by slow liquid flow rates in these nano-devices. The sensitivity of protein sequencing will also require further creative input. In this area, interfacing the microfabricated array to the mass spectrometer will need particular attention.

A further technology that is expected to improve MS sensitivity is the development of the highly sensitive electrospray time of flight (ESI-TOF-MS), electrospray quadrupole time of flight mass spectrometers (ESI-QTOF-MS), and quadrupole ion trap coupled with a reflecting time-of-flight instrument. The development of such instruments is well underway. In addition to high sensitivity, these instruments have a short duty cycle, which enables very fast data acquisition, with no loss of sensitivity. These instruments also provide a constant medium resolution (>5000 full width half maximum [fwhm] definition), and good mass accuracy. The use of isotopic labeling (as achieved when enzymatic protein digests are carried out in 18O water) can aid peptide identification since MS/MS instruments allows 18O enriched ions to be readily identified in spectra, facilitating peptide sequence identification.

In summary, it is clear that there will need to be a significant pursuit of more sensitivity for CE-MS. In this regard, MFD-MS may, with further development and possible coupling to a highly sensitive mass spectrometer (such as an ESI-TOF-MS), provide the technology that is needed for future generations of proteomic research. However, lessons learned in the macro world of CE-MS will need to be heeded as new MFDs and MS interfacing techniques are developed. In particular, input by material scientists may provide improved surfaces to prevent interaction and loss of peptides and proteins within the channels of MFDs. Assuming that these challenges will be overcome, technology for isolation, manipulation, digestion, and sequencing extremely low levels of biologically active peptides and proteins will become a reality within the next few years.

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