METHODS AND MATERIALS

Materials

Ammonium bicarbonate, ethylenediaminetetraacetic acid (EDTA), formic acid, methanol, sodium phosphate and urea were obtained from Merck (Darmstadt, Germany). Dithiothreitol (DTT), iodoacetic acid (IAA), and iodoacetamide (IAM) were obtained from Sigma (St Louis, MO, USA). Bovine serum albumin (BSA) was obtained from ICPBio, Auckland, NZ). Acrylamide was obtained from Bio-Rad (Hercules, CA, USA). LC/MS-grade acetonitrile was obtained from Fisher Scientific (Pittsburg, USA). TPCK-trypsin was obtained from Promega (Fitchburg, MI, USA). LC/MS-grade water was obtained from Fluka. Empore disks were obtained from Supelco (Bellefonte, PA, USA).

Preparation of merino wool

Merino wool with fibers of ~18 μm diameter were sourced from a ewe from a farm in mid-Canterbury, New Zealand. The wool was detipped by removing the top third of the fiber and the extraneous dirt and sebum removed by subjection to agitation in an ultrasonic bath in ultrapure water for 15 mins three times. After this the wool fibers were washed sequentially twice in dichloromethane, twice in ethanol, then twice in water before air drying overnight. After this the wool was cut into snippets of less than 1 mm length with scissors.

Sequential alkylation with IAM, acrylamide and IAA

The method of stepwise reduction and labelling of cysteines is summarized in Figure 1, where cysteines in the wool fiber are progressively reduced by increasing the concentration of DTT. This experiment was split into three steps involving the reduction of five independent samples with 5 mM, 10 mM and 15 mM of DTT, followed by alkylation with one of three different reagents: iodoacetamide (IAM), acrylamide and iodoacetic acid (IAA) to determine at which step the cysteine group became accessible to the labelling reagent.
Prior to commencing the experiment, a control run was made with a sample of the snipped wool in the absence of DTT. This was incubated in the base buffer solution only (100 mM sodium phosphate buffer (pH 7.4), 1 mM EDTA) for 24 h followed by labelling with 2 mM IAM in base buffer for 30 min in the dark. The supernatant was collected after the extract was centrifuged at 44,000 g for 15 min. This was Step 0 (Figure 1).
In the first step, 20 mg of cleaned wool was incubated in 1.5 mL of the base buffer containing 5 mM DTT for 24 h followed by labelling with 2 mM IAM in the base buffer for 30 min in the dark. The supernatant was collected after the extract was centrifuged at 44,000 g for 15 min. Residual extraction buffer was removed from the pellet by vortexing for 1 min in wash buffer (10 mM sodium phosphate buffer (pH 7.4), 1 mM EDTA), followed by centrifugation at 44,000 g for 15 min. This was repeated three times and these washes discarded. After this the pellet was suspended in wash buffer and centrifuged. Mass spectrometry was used to detect the presence of residual IAM. This was performed by taking 60 µL of the supernatant from the washed wool pellet and added to 10 µL of 0.2 mg/mL of reduced BSA and vortexed in the dark for 30 min at room temperature. This BSA was digested with trypsin and the resulting digest analyzed for alkylation of the BSA peptides by mass spectrometry. If no alkylation was detected in the BSA peptides, indicating the absence of residual IAM, the washed pellet was then used in the next step for further analysis.
In the second step the washed wool pellet was incubated in the base buffer solution containing 10 mM DTT for 24 h. This was followed by labelling with 350 mM acrylamide in the base buffer for 24 h in the dark. The supernatant was then collected after centrifugation and the pellet washed three times with wash buffer and checked for residual alkylating reagent, as described above, before proceeding to the next step.
In the third step the washed wool pellet was incubated in the base buffer containing 15 mM DTT for 24 h. This was followed by labelling with 2 mM iodoacetic acid in base buffer for 30 min in the dark, after which the extract was centrifuged and the supernatant collected, while the pellet was discarded.

Preparation for Mass Spectrometry

Each extract was digested with TPCK-trypsin at an enzyme-substrate ratio of 1:50 in 50 mM ammonium bicarbonate:AcN (9:1) at 37 °C for 18 h. The peptide digest was vortexed for 3 h in the presence of Empore™ disks that had been prewetted with AcN and methanol. Following this the Empore™ disks were washed with 0.1% TFA and extracted with 75% AcN in 0.1% trifluoroacetic acid. After drying the extracts were resuspended in 50 μL of 0.1% trifluoroacetic acid for further analysis.

Mass spectrometry analysis

LC-MS/MS was performed on a nanoflow UPLC system (Dionex Ultimate 3000) coupled to an Impact II mass spectrometer equipped with a CaptiveSpray source (Bruker Daltonik, Bremen, Germany). For each sample, 1 μL of the sample was loaded on a C18 PepMap100 nano-Trap column (200 μm x 2 cm) at a flow rate of 5 μL/min. The trap column was then switched in line with the analytical column ProntoSIL C18AQ (100 μm ID x 150 mm 3 micron 200 Å). The reverse phase elution gradient was from 2% to 20% to 45% B over 60 min, total 84 min at a flow rate of 1000 nL/min. Solvent A was LCMS-grade water with 0.1% formic acid; solvent B was LC-MS-grade AcN with 0.1% formic acid.
Samples were measured in automatic MS/MS mode, with a mass range of m/z 150–2200. Acquisition speed was 5 Hz in MS and 1-50 Hz in MS/MS mode depending on precursor intensity. Ten precursors were selected in the m/z 300-1250 range, with a preference for doubly or triply charged peptides. The analysis was performed in positive ionization mode with a dynamic exclusion of 60 sec.

Data Analysis

Following the LC-MS run, the QTOF data were searched using Peaks Studio 8.5 (Bioinformatics Solutions Inc). The raw data were refined by a built-in algorithm and protein/peptides were identified using the following parameters: a precursor mass tolerance of 10 ppm; fragment mass tolerance of 0.05 Da, a taxonomy of Ovis aries with an in house Sheep_uniprot_ovine_2016_10_plus_keratins database. This database was derived from the Uniprot 2016_10 database, augmented by 54 AgResearch sequences from EST contigs, annotated by BLAST searching against the NCBI database and keratin sequences from the NCBInr non-redundant database. Trypsin was chosen as the proteolytic enzyme and up to 2 mis-cleavages were allowed. Carbamidomethyl (C), propionamide (C) and carboxymethylation (C) were chosen as variable modifications. A false discovery rate (FDR) of 0.5% was used for peptide identification in Peaks. In addition, the Peaks post-translational modification (PTM) A-score was set to 30, de novo only ALC > 85% and only proteins with a minimum of 2 unique peptides identification were included. As the keratin-family of proteins is known to have highly similarly in protein sequences, the quality of identification of all peptides was checked manually.
In the final analysis a peptide sequence containing the same labelled cysteine was only considered to be significant if it was found in four of the five repeats. This was because in our previous study it was established that only peptide sequences with the same labelled cysteine observed in at least four out of five repeats had a significance level of P <0.05 12. That is, any peptide with less than four observations was considered to have non-repeatable, and therefore random, accessibility.