Mass Spectrometry in Systems Biology - Systems Biology: Networks, Models, and Applications - page 11

expected outcome. This chapter is centered on one aspect of large-scale
analysis by MS, tandem MS or sequencing of biomolecules by MS/MS.
Sequencing of Bioanalytes by Mass Spectrometry
Mass measurement alone does not elucidate the covalent structure
of ions, particularly for peptides and proteins, thus complete physico-
chemical characterization of a bioanalyte requires additional analysis.
Tandem MS analyzes complex mixtures of biomolecules by first select-
ing a precursor ion for fragmentation to obtain an MS/MS spectrum
that contains a specific signature (pattern) for each biomolecule. We
compiled below the nomenclature and salient features of MS/MS
(sequencing) spectra of peptides and lipids. Sequencing spectra together
with appropriate multistage MS experiments could describe the struc-
ture as well as the profile changes of biomolecules.
MS/MS Spectra of Peptides . Fragmentation of peptide bonds produces
two major types of ions: b ions (charge on peptide N-terminus) and
y ions (charge on peptide C-terminus) [17]. MS/MS spectra of peptides
are readouts of their amino acid sequence. The appearance of a peptide
MS/MS spectrum resembles a histogram, where the bars (fragment ions)
often represent differences corresponding to the molecular weight of an
amino acid. Database searching of theoretical spectra or library match-
ing of previously collected MS/MS spectra identifies the peptides [18].
Large-scale analysis of proteins involves searching hundreds of thou-
sands of MS/MS spectra. Consequently, results of database searches
should be statistically evaluated in order to provide cutoff filters for
protein identification [19,20].
Peptides are polymers of repeating monomeric units. Fragmentation
along the backbone of the peptide produces a mass ladder, that is, a
double fingerprint that sometimes is complemented with fragmenta-
tion of chemical units branching from the backbone (figure 1.4).
MS/MS Spectra of Lipids. Annotation of lipid MS/MS spectra is given
by XX:Y (XX is the total number of carbons of the fatty acids, Y being
included on an exclusion list for limited duration. Therefore, after evaluating
scan #2761, the instrument proceeds with MS/MS sequencing of the ions
that are not already in the exclusion list. Consequently, the fragmentation
spectra of m / z 509.2, 1050.9, and 701.3 are recorded in scan #2762 (c), 2763 (d),
and 2764 (e), respectively. Starting with scan #2765 the instrument repeats the
cycle of acquisition. Using the Sequest database search algorithm, the following
peptide sequences were identified: #2762 KIKVYLPR (doubly charged) from
ovalbumin, #2763 LKEC am C am DKPLLEKSHC am IA (doubly charged) from
albumin, #2764 LKEC am C am DKPLLEKSHC am IA (triply charged) from albumin.
Cysteines present in the albumin peptide are carbamidomethylated.