(MS/MS) techniques. Typically, the MALDI-TOF MStechniques are used to quickly identify peptide fragmentsand conﬁrm the presence of known proteins. Nano-scalecapillary LC/MS/MS techniques (using 50–100
mdiameter columns, operating at ﬂow rates of 20–500nL/min) are used to further interrogate the complex proteinmixture at the low femtomole level. These techniquesrequire the use of a specialized ESI source shown in Fig. 3
.The combination of MALDI-TOF MS and capillaryLC/MS/MS was recently described for the identiﬁcation of disease state markers in human urine (12). In this study,urine proteins obtained from emphysema patients wereseparated on 2-D gels and selected spots were digestedwith trypsin and analyzed by MALDI-TOF. A databasesearch using Protein Prospector identiﬁed a potentialbiomarker for emphysema as human alpha-1-antitrypsin(A1AT). The corresponding MALDI spectrum containednine out of 18 peptides with masses that match theexpected tryptic digest fragments for A1AT.The same tryptic digest protein sample was analyzedby capillary LC/MS/MS using an ion trap massspectrometer followed by a database search withSEQUEST.
Fig. 4 illustrates the components of an iontrap mass spectrometer. The highly automated data-dependent MS/MS analysis provided excellentsequence coverage for 11 tryptic peptides related toA1AT in a single LC/MS run. A tryptic peptide thatcorresponds to the A1AT sequence SVLGQLGITK observed at retention time (
) 30.5 min. was observedin the spectrum. The LC/MS data also providedsequence information on unmatched MALDI peaks.The need to detect lower concentration of protein andpeptide mixtures has resulted in the increased use of hybrid quadrupole/orthogonal TOF (QTOF) MS/MSinstruments (13) in conjunction with microcolumn LC.Fig. 5
shows a schematic of a QTOF instrument. ThisLC/MS approach provides a resolution of ca. 0.1 massunits allowing for the analysis of complex product ionspectra (14,15). A recent publication by Chalmers andGaskell highlights the current challenges in proteomeanalysis with regard to MS instrumentation (16).
NATURAL PRODUCTS DEREPLICATION
Historically, an excellent source of novel lead drugcompounds is natural products. Natural product screeningactivities typically occur during drug discovery and
Schematic of a nano-scale capillary ESI interface. This specialized LC/MSinterface, operating at ﬂow rates from 20 to 500 nL/min and using 50 to 100
m IDcolumns, typically provides low femtomole sensitivity. Fully automated samplehandling and preparation procedures (i.e., desalting and preconcentration) combinedwith specialized devices for high separation and variable nL gradient ﬂow ratesprovide unique capabilities for high-throughput analysis of proteins. (Courtesy of New Objective, Cambridge, MA.)
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