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Question Set 1: Peptides that kill pain [20 points] During your investigations of an arthropod, you clone a cDNA coding for a 34residue polypeptide.

Question Set 1: Peptides that kill pain [20 points]

During your investigations of an arthropod, you clone a cDNA coding for a 34‐residue
polypeptide. This polypeptide, which is suspected to have analgesic properties, has the following
deduced sequence:


With the objective of learning more about this polypeptide in its native condition, you raise
polyclonal antibodies against a synthetic peptide with the amino acid sequence
. In so doing, you succeed in immunoaffinity purifying the
native polypeptide from the arthropod from which the cDNA was originally cloned. As much as
you would like to sequence the purified native polypeptide in its entirety by the Edman
degradation using an automated protein sequenator, the availability of the source arthropod and
the yield of the native polypeptide purified by immunoaffinity chromatography are such that you
have only sufficient material for its analysis by MALDI‐TOF MS. However, when it comes to
analyzing the purified native polypeptide by MALDI‐TOF MS you obtain unexpected results.
That is, when you subject an aliquot of the purified preparation, which contains only one
polypeptide, to exhaustive digestion with trypsin, you obtain the m/z species listed in Table 1

Table 1: m/z listing of five peptide ions in tryptic digest of native polypeptide

Peptide ion m/z (tolerance ± 0.01)
1 232.1404
2 288.2030
3 474.2823
4 1366.6288
5 2058.8295

Note that the m/z values of these peptide ions were generated following a standard protocol:
Aliquots of the immunoaffinity purified native polypeptide were digested with 6 µg/ml trypsin
(sequencing grade, modified) for 7 h, after which time the digest was successively extracted with
0.1% (v/v) formic acid in 2% (v/v) acetonitrile and with 0.1% (v/v) formic acid in 50% (v/v)
acetonitrile. For mass analysis in a Micromass MALDI Reflectron MS (Waters/Micromass), 1 µl
aliquots of the tryptic digest extract were mixed with 1 µl 4 mg/ml matrix
(α‐cyano‐4‐hydroxycinnamic acid) dissolved in 0.1% (w/v) trifluoroacetic acid/50% (v/v)
acetonitrile before spotting 1 µl aliquots onto a stainless steel target plate. The MALDI‐TOF MS
instrument which was equipped with a pulsed nitrogen laser (337 nm, pulse width 4 ns) was
operated in reflectron mode in the mass range 200‐3500 Da at an accelerating voltage of 20 kV.
After combining the spectra from 200 laser shots at several positions in the target spot, five
peptide ions were identified at high confidence.


A. How do you interpret these results? What is the most straightforward explanation for
the m/z species identified? (Hint: Assume that the m/z values listed in Table 1 are those
of the monoisotopic species and that the successive isotopologs in the isotope envelopes
each of the m/z species listed are separated from each other by 1 atomic mass unit
[amu]. Also assume that the five m/z species listed in Table 1 were the only species
identified in the MALDI-TOF MS). [8 points]

B. Explain any ambiguities that arise in your interpretation of these results. For example,
are there alternate straightforward explanations that are equally consistent with the
results (i.e., capable of yielding precisely the same results numerically)? [4 points]

If you were to isolate a naturally occurring mutant of this polypeptide which has a Ser
residue instead of a Cys residue at position 17, and this mutant was to yield the monoisotopic
m/z listing shown in Table 3 upon tryptic digestion under the same conditions as those used to
digest the wild type native polypeptide in Table 2.

C. How would the results in Table 2 affect your conclusions? (Hint: Assume that the wild
type and C17S mutant polypeptides are otherwise identical except for the Cys to Ser
substitution and its consequences). [4 points]

Table 2: m/z listing of six peptide ions in tryptic digest of C17S mutant polypeptide

Peptide ion m/z (tolerance ± 0.01)
A 232.1404
B 288.2030
C 474.2823
D 822.3603
E 1223.5149
F 1366.6288

In framing this question, it was stated that the cDNA coding for the wild type 34‐residue
polypeptide, which is suspected of having “analgesic properties”, was isolated from an

D. On the basis of what you now know of the polypeptide, are there any other polypeptides
like it that have already been discovered? Are there any known homologs (think
BLAST!), albeit distantly related ones, from the same phylum that have similar
properties? If so, would you be justified in thinking that the polypeptide you have
discovered might find application in the treatment of muscular dystrophy, neuropathic
pain and/or atrial fibrillation? [4 points]

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