Biology Reference
In-Depth Information
peptide extracts, and sequenced by Edman degradation.
30,70
e
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Although
effective, this approach required large amounts of material (e.g. from
60,000 worms) and extensive and time-consuming purification proce-
dures. Since then, expressed sequence tag (EST) and genomic sequences
have led to the prediction of precursor proteins that include many more
putative peptides, including more FLPs,
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and the NLPs which may or
may not have C-terminal amidation.
74
Since the precursor proteins are
cleaved by proteases in the secretory pathway, and different cells express
different proteases, it is important also to detect the peptide product itself,
since predictions from the common cleavage sites (typically single or
pairs of basic amino acids) may be erroneous. Alternative splicing is also
an issue that affects the interpretation of peptide-encoding genes found in
genomic DNA.
Peptide discovery has recently been vastly aided by the use of mass
spectrometry (MS). Initially, matrix assisted laser desorption ionization-
time of flight (MALDI-TOF) MS was used on dissected ganglia to detect
peptides from single ganglia.
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In this technique, there is the simultaneous
vaporization and ionization of the molecules in the sample; the released
ions are then accelerated by a voltage gradient and the time taken to drift
down a flight tube to a target that detects each ion is proportional to the
momentum of the ion, which is in turn dependent on the mass to charge
ratio. Tandem MS, in which selected ions are fragmented, also allows
de novo sequencing, which can also be carried out on the peptides from
single ganglia. In other systems, it was already possible to carry out MS on
single neurons,
76,77
but in A. suum, the isolation of single neurons was
difficult because of their tight association with surrounding hypodermal
tissue, which acts as the glia (the non-neuronal tissue that surrounds
neurons in most higher organisms) in nematode nervous systems. Finally,
the dissection technique for isolating single neurons was perfected, and
subsequent MS and MS/MS together facilitated the discovery of many
novel peptides.
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About 25% of the different types of neurons in A. suum
have been examined so far, and all contain neuropeptides; when the
transcripts encoding these peptides are examined, often it is clear that the
peptides in a single neuron are the products of multiple genes. Many of
the peptides are novel, and in many cases the previously unknown
peptides outnumber the known peptides. One of the additional advan-
tages of single cell MS is that besides the chemical identification of the
processed peptides in a neuron, the cellular localization is simultaneously
described. This is usually extended and validated by other techniques
such as immunocytochemistry (if specific antibodies can be generated)
and/or in situ hybridization, so that the full expression pattern of
peptides and their encoding transcripts can be determined. With very rare
exceptions, the three techniques give identical results. Although none of
these three techniques is quantitative, there are consistent differences in
