Dried peptides were dissolved in 0.5% (vol/vol) heptafluorobutyric acid and 1% (vol/vol) formic acid (FA) and were separated by nano-LC using an HPLC (Ultimate 3000; Dionex, Amsterdam, Netherlands) and autosampler system (Dionex). Peptides (5 μL) were loaded onto a microcolumn (0.76 mm × ∼15 mm; SCX; Dionex) containing HPLC media (Poros S10; Applied Biosystems, Foster City, CA) and eluted sequentially using 5, 10, 15, 20, 25, 30, 40, 50, 100, 250, 500, and 1000 mM ammonium acetate (20 μL). The initial unbound fraction and each of the salt steps were concentrated and desalted on a microprecolumn (500 μm × 2 mm; C18; Michrom Bioresources, Auburn, CA) with 98% (vol/vol) water, 2% (vol/vol) acetonitrile, and 0.1% (vol/vol) FA (buffer A) at 20 μL/min. After a 4-minute wash, the precolumn was switched (Valco 10 port valve; Dionex) into line with a fritless nanocolumn (75 μm × ∼10 cm) containing C18 media (5 μm, 200 Å Magic; Michrom Bioresources). Peptides were eluted using a linear gradient of H2O/CH3CN (98:2, 0.1% [vol/vol] FA) to H2O/CH3CN (55:45, 0.1% [vol/vol] FA) at 350 nL/min over 30 minutes. High voltage (1800 V) was applied to a low-volume tee (Upchurch Scientific, Oak Harbor, WA), and the column tip was positioned approximately 0.5 cm from the heated capillary (T = 200°C) of a mass spectrometer (LTQ FT Ultra; Thermo Electron, Bremen, Germany). Positive ions were generated by electrospray, and the mass spectrometer (LTQ FT Ultra; Thermo Electron) was operated in data-dependent acquisition mode. Each sample was analyzed by LC/MS once.
A survey scan (m/z 350-1750) was acquired in the Fourier transform ion cyclotron resonance cell (resolution = 100,000 at m/z 400, with an initial accumulation target value of 1,000,000 ions in the linear ion trap). As many as seven of the most abundant doubly or triply charged ions (>2000 counts) were sequentially isolated and fragmented within the linear ion trap using collision-induced dissociation, with an activation
q = 0.25 and an activation time of 30 ms at a target value of 30,000 ions. The m/z ions selected for MS/MS were dynamically excluded for 60 seconds. All data were acquired in profile mode and searched against the SWISS-PROT 54.3 database (17,400 human sequences; made available by Swiss Institute of Bioinformatics) with a range of posttranslational modifications (PTMs) using MASCOT (version 2.2.03; Matrix Science, London, UK), with the enzyme specificity set to semi-trypsin. The numbers of MS/MS spectra searched were as follows: fetal, 12,585 and 14,262; normal 16,186, 10,011, and 16,156; cataract, 12,120, 14,378, and 17,761. The following PTMs were listed as variable modifications in addition to deamidation: acetylation (N-term), Arg55, a newly noted unknown modification to Arg
31 ; carboxymethyl,
N-ethylmaleimide, methylation (C); oxidation (H, W, M); peptide tolerance, 8 ppm; fragment tolerance, 0.6 Da; one missed cleavage allowed. MASCOT results were processed with the Trans-Proteomic Pipeline (version 3.2.0); a global false-discovery rate of
P < 0.05 was chosen using PeptideProphet statistics once the MASCOT search was fully processed by this algorithm.