Mass spectrometric identification and relative quantitation of chicken lens crystallins

Soluble proteins were isolated from 7–8 week-old freshly dissected chicken lenses after overnight shipping on ice (Pel-Freez, Rogers, AK). A total of 6 lenses ranging in wet weight between 51 and 68 mg were homogenized in 1.0 ml of 20 mM phosphate buffer (pH 7.0), and 1.0mM EGTA and centrifugated at 16 000 × g for 15min. The insoluble pellet was then resuspended in 1 ml of homogenizing buffer, centrifuged again, and the soluble fraction added to the initial supernatant. This was repeated a total of 3 times. The pellet was then suspended with a probe sonicator using 3 × 5 sec burst on ice, and the protein content determined using a BCA assay and bovine serum albumin as a standard (Thermo Scientific). 100 μg portions of soluble and insoluble fractions were then dried by vacuum centrifugation, resuspended in digestion buffer containing 8 M urea, proteins reduced/alkylated, and digested overnight with trypsinas previous [52]. Peptides were then injected onto a NanoEase XBridge BEH130 5 μm particle 0.3 × 50 mm C18 column (Waters) using a mobile phase containing 10 mM ammonium formate (pH 10) with a 3 μl/min flow rate. Peptides were than eluted by sequential injection of 20 μl volumes of the same mobile phase, but containing 14, 20, 22, 24, 26, 28, 30, 40, and 90% acetonitrile. Eluted peptides were then diluted at a tee with a mobile phase containing 0.1% formic acid at a flow rate of 24 μl/min to acidify samples and dilute acetonitrile concentrations. Peptides were then separated by second dimension reverse phase C18 chromatography at low pH by trapping peptides on an Acclaim PepMap 0.1 × 20 mm NanoViper C18 peptide trap (Thermo Scientific), then separation using a PepMap RSLC C18, 2 μm particle, 75 μm × 25 cm EasySpray column (Thermo Scientific) using a 7.5 –30% acetonitrile gradient over 90 min in a mobile phase containing 0.1% formic acid and a 300 nl/min flow rate using a Dionex NCS-3500RS UltiMate RSLCnano UPLC system. Tandem mass spectrometry data was collected using an Orbitrap Fusion Tribrid mass spectrometer configured with an EasySpray NanoSource (Thermo Scientific). Survey scans were performed in the Orbitrap mass analyzer at 120,000 resolution, data-dependent MS2 scans in the linear ion trap collected in top speed mode with dynamic exclusion enabled and using higher energy induced dissociation following isolation using the instrument’s quadrupole. Further details of instrument settings are found in Supplementary Materials.

Peak lists in MS2 format [53] were created using MSConvert (ProteoWizard version 3.0.6705) [54] and in-house scripts. Peptides were identified using Comet [55] version 2014.2 rev. 2. Searches used either a Chicken Ensembl v77 protein database (16,366 sequences) or a UniProt Gallus gallus reference proteome (17,623 sequences downloaded 12/12/2014 from www.uniprot.org). Databases also included contaminants and reversed decoy sequences. Ensembl databases were reformatted to include protein descriptions by using information from BioMart and in-house scripts. Search parameters were: 2.5 Da average parent ion tolerance, 1.0005 Da monoisotopic fragment ion tolerance, variable oxidation of methionine (M+15.994), fixed alkylation of cysteine (C+57.02), and trypsin enzymatic cleavage with a maximum of 2 missed cleavages. Peptides were filtered to 1% false discovery rates (FDR) using the PAW pipeline[34]. Identified proteins were matched between the Ensembl results and the Uniprot results using a local installation of BLAST [56, 57] and in -house scripts. Some minor crystallins were not well annotated in Uniprot and lens fiber major intrinsic protein (MIP26) was missing from Ensembl.

Proteins were inferred from peptides using parsimony principles where identical peptide sets were reported as protein groups and peptide subsets were not reported. A minimum of two distinct peptides per protein was required for protein identification. An additional processing step used extended parsimony principles to group highly homologous proteins into families. For water-insoluble proteins, there were 1705 identified protein groups (0.6% FDR). Due to the relatively higher crystallin abundances, only 1039 protein groups (0.5% FDR) were identified for the water-soluble fraction. Relative crystallin abundances within each solubility fraction were estimated using MS2-intensity-weighted spectral counts, which have been shown to be more accurate than simple spectral counts [35]. Complete instrument settings, search parameters, and pipeline processing steps are included in the Supplemental data along with complete lists of identified peptides, identified proteins, and quantitative information.