Tandem mass spectra were acquired using a quadrupole time-of-flight (QTOF) Ultima mass spectrometer with a nanospray source (Waters/Micromass, Manchester, UK). Digested peptides were separated by 1D LC using a NanoCapLC autosampler system (Waters, Milford, MA) and the system was comprised of three pumps (pump A, B, and C), an autosampler, a sample cooler, and a switch valve. A partial loop injection method was used; 2 μL of the sample was picked up with a flush volume of 4 μL. After 20 minutes of sample loading onto the analytical column, with a flow rate of ∼350 nL/min (mobile phase A from pump A (H
2O:FA (99.9:0.1, vol/vol), the 10-port switch valve (Valco, Houston, TX) was automatically switched into line with buffers to bypass the injection valve and loop. Peptides were eluted with acetonitrile in mobile phase B (from pump B, H
2O:ACN:FA (19.9:80:0.1, vol/vol). The separation of the peptides was performed on a fritless analytical Magic C18 reversed phase column with 3 μm packing material (Michrom Bioresources, Auburn, CA) particle size 200 Å; 75 μm inner diameter; 360 μm outer diameter and ∼20 cm long, manufactured according to Gatlin.
32 Two different gradients were optimized for human αA and γS crystallin, and peptides were eluted with a flow rate of ∼200 nL/min over 80 minutes. For αA crystallin a linear gradient of 10% to 17% mobile phase B (vol/vol) was used, while that for γS crystallin was a linear gradient of 19% (vol/vol) to 28% (vol/vol) mobile phase B. The analytical column was connected via a fused silica capillary (∼20 cm; inner diameter = 25 μm, outer diameter = 360 μm) to a low volume tee (Upchurch Scientific, Silsden, UK) where a high voltage (2400 V) was applied to the capillary and the column tip positioned ∼0.5 cm from the Z-spray inlet of a QTOF tandem mass spectrometer. The mass spectrometer was operated in positive ion mode, with a source temperature of 80° C and a cone voltage of 50 V. The QTOF was operated in data-dependent acquisition mode (DDA), with an acquisition of TOF MS survey scan (m/z 350–1700, 1 second) and the two largest multiply charged ions (counts > 30) were sequentially selected by Q1 for MS/MS analysis. Argon was used as collision gas, and optimum collision energy would be chosen based on its charge state and mass. Tandem mass spectra were accumulated for up to 2 seconds (m/z 50–2000). Individual fragment spectra obtained for each of the precursors were processed using MassLynx software version 4.0 SP4 (Waters/Micromass) to obtain centroid MS/MS data and the corresponding peak lists in the format of pkl files.