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where one of the peptides separated for mass-fingerprinting is further fragmented in

a second analyser, and from the fragmentation pattern sequence data can be deduced

(mass spectrometry conveniently fragments peptides at the peptide bond, such that

the difference in the mass of fragments produced can be related to the loss of specific

amino acids; Section 9.5.2). This partial sequence data is then used to search the protein

sequence databases for sequence identity. Universal databases are available that store

information on all types of protein from all biological species. These databases can be

divided into two categories: (i) databases that are a simple repository of sequence

data, mostly deduced directly from DNA sequences, for example the Tr EMBL data-

base; and (ii) annotated databases where information in addition to the sequence

is extracted by the biologist (the annotator) from the literature, review article, etc., for

example the SWISS-PROT database.

An example of how sequence data can be produced is shown in Fig. 8.6. A lysate of

2  106 rat basophil leukaemic (RBL) cells were separated by 2-D electrophoresis and

spot 2 chosen for analysis. This spot was digestedin situusing trypsin and the

resultant peptides extracted. This sample was then analysed by tandem MS using a

triple quadrupole instrument (ESI-MS^2 ). MS of the peptide mixture showed a number

of molecular ions relating to peptides. One of these (m/z890) was selected for further

analysis, being further fragmented in a quadrupole mass spectrometer to give frag-

ment ions ranging fromm/z595.8 to 1553.6 (Fig. 8.6). The ions atm/z1002.0, 1116.8,

1280.0, 1466.2 and 1553.6 are likely to be part of a Y ion series (see Fig. 8.6) as they

appear at higherm/zthan the precursor atm/z890. The gap between adjacent Y ions is

Tag is

(1116.8) YWS (1553.6)

300

1000

600 900 1200 1500 m/z

10000

15000

1280.0

1116.8

1002.0

758.8890.2

595.8

1553.6

YENDY

WS

5000

Fig. 8.6Nano-ESI MS^2 spectrum ofm/z890 from RBL spot 2 showing construction of a sequence tag. They-axis

shows relative intensity. (Courtesy of Glaxo SmithKline, Stevenage, UK.)

343 8.5 Proteomics and protein function