RESULTS
Sequential labelling of
cysteines
In order to determine the relative accessibility of cysteines in wool
the keratins and KAPs were subjected to a process of sequential
alkylation as the concentration of DTT was raised in stepwise fashion
from 0 to 15 mM.
A total of 38 peptides were detected and identified in the mass spectrum
of protein extracts in the control run, involving wool that had not been
treated with a reductant. Of these, two were from the epithelial keratin
K10 and the rest were from either actin or serum albumin. A number of
cysteines in the peptides were labelled with IAM but only in those from
serum albumin.
In contrast cysteine labelling of keratins and KAPs was only detected
when the wool was incubated with DTT. Among the labelled cysteines were
a number identified in our earlier study 12,
specifically: in the tail domain of K31, T-42 and T-45; in coil 1B of
K31 and K33b, 1B-87; the head domain of K35, H-36; H-24 and H25 in K81
(H-29 and H30 in K83); and H61 and T-24 in K85.
In the first step, when a DTT concentration of 5 mM was used to reduce
the proteins, eight peptides from four keratins and three KAPs
containing IAM-labelled cysteines were identified in at least four out
of the five biological repeats (Figures 2-4, Table 1). Interestingly,
all the keratin peptides identified were labelled on either the head or
tail domain. In the case of the type I keratins one peptide from the
tail region of K31 was identified on which four cysteine residues were
labelled. Peptides from three Type II keratins were also found to have
IAM-labelled cysteines. For K83 this involved two peptides from the head
domain of which three cysteines were found to be labelled. In the case
of K85 a peptide containing one cysteine from its tail domain was
labelled, while a peptide containing one cysteine from the tail domain
of K86 was also labelled. IAM-labelled peptides were identified from
three HSPs, specifically cysteines close to the N-terminus of KAP2.3 and
the cysteines close to the C-terminus of KAP7.1, KAP11.1 and KAP13.1
(Figure 2, Table 1).
In the second step, proteins were unraveled further by reduction with 10
mM DTT and then labelled with acrylamide (Figures 2-4, Table 2). This
resulted in the identification of a total of 62 cysteine-labelled
peptides, of which 20 were from keratins and 42 from KAPs. Of the KAPs,
65% of were classed as HSPs, 16% UHSPs and the rest high
glycine-tyrosine proteins (HGTPs). In the case of the type II keratins
all the newly characterized peptides in this step (and hence cysteines)
were either located in the head or tail domains (Figure 4). In contrast,
in the type I keratins one acrylamide-labelled cysteine was identified
in a peptide from coil 1B and another from coil 2, while an
acrylamide-labelled cysteine was identified in a peptide from the L1
linker in K34 (Figure 3). No modified cysteines were found in the
hendecad regions 3. As found previously12 around 90% of the labelled cysteines in the HSPs
and UHSPs are found close to a proline residue in the sequence and over
half within 20 residues of the N- and C-terminus of the proteins.
Specifically these involved cysteines at the N-terminus of KAP3.3,
KAP3.4 and KAP7.1, and cysteines close to the C-terminus of KAP31,
KAP3.4, KAP4.2, KAP4.5, KAP9.2, KAP9.8, KAP10.11, KAP10.12, KAP12.2,
KAP19.5 and KAP26.1 (Figure 2, Table 2). At the same time a number of
cysteines were detected from the more central regions of some of the
KAPs, among them KAP2.3, KAP3.4, KAP4.2, KAP6.1, KAP6.4, KAP10.4,
KAP11.1,KAP13.1, KAP16.1-like, KAP19.3, KAP24.1 and KAP26.1.
In the third step very few IAA-labelled peptides were identified in the
final alkylation step. Of the two keratin peptides identified the one
from K31 was labelled in the coil 2 domain while that from K35 was
labelled in the head domain (Figure 3, Table 3). Two KAPs were
identified in this stage, one being KAP 6.1, an HGTP, and the second
being from the HSP KAP13.1-like (Figure 2, Table 3).
Validation of the labelling
experiments
To determine if the large increase in peptides identified in step 2 and
the subsequent drop in peptide numbers in step 3 was due to the longer
labelling time used with acrylamide the entire process was repeated
using IAM in all steps. However, a similar pattern was observed when
comparing the proportion of peptides identified between each of the
steps. This led to the conclusion that the alkylation reagent used was
not influencing the efficiency of the labelling and thus peptide
identification.