Tissue Proteomics Workflow for LC-MS/MS

Extracts taken from tissue sections were pipetted directly into 3µL of the extraction solvent. Aliquots of 1–2µL, containing 25–50% of each tissue extract, were diluted 6-fold with 0.1% formic acid to reduce the organic composition in the solvent to 3% acetonitrile. For lens extract analysis, 1 µL (25%) of the tissue extract was used, and 2-µL aliquots (50%) of the kidney and brain extracts were analyzed by LC-MS/MS. The diluted protein extracts were loaded onto a reverse-phase capillary trap column using a helium-pressurized cell (pressure bomb). The trapping column (360µm × 150µm ID) was fritted with a filter end-fitting (IDEX Health & Science), and was packed with 5 cm of C₈ material (15 µm material, 300Å Grace Vydac or 5µm, 300 Å YMC Co.). Once the sample was loaded, an M-520 microfilter union (IDEX Health & Science) was used to connect the trap column to an analytical column (360µm × 100µm ID, 8 cm or 20 cm of C₈ stationary phase material), equipped with a laser-pulled emitter tip. Using an Eksigent NanoLC Ultra HPLC, peptides were gradient-eluted at a flow rate of 400 nL/min, and the mobile phase solvents consisted of 0.1% formic acid, 99.9% water (solvent A) and 0.1% formic acid, 99.9% acetonitrile (solvent B). The gradient consisted of 2–60 %B in 50 min, 60–90 %B in 10 min, 90 %B for 1 min, 90-2 %B in 1 min, and the column was equilibrated at 2 %B for 12 min. Upon gradient-elution, proteins were mass analyzed on a LTQ Velos mass spectrometer, equipped with a nanoelectrospray ionization source (Thermo Scientific). The instrument was operated using a data-dependent method or a combination method consisting of both data-dependent and targeted scan events. Dynamic exclusion was enabled allowing a repeat count of 4 within 15 s. For all LC-MS/MS analyses, full scan (m/z 360–2000) spectra were acquired as the initial scan event, and the seven or eight most abundant ions in each MS scan were selected for fragmentation in the LTQ. For electron transfer dissociation (ETD) of eluting protein species, an isolation width of 2.5 m/z and reaction times of 90–115 ms were used. The MSⁿ AGC target value in the ion trap was set to 1e⁴, with a maximum injection time of 100 ms. The ETD reagent ion AGC target was 3e⁵ or 5e⁵ with a maximum inject time of 100 ms. For a subset of LC-MS/MS analyses, a data-dependent ETD/CID toggle method was used, where sequential ETD and CID spectra were acquired of the same protein precursors in back-to-back scan events. ETD and CID MS/MS spectra were interpreted manually with assistance by the TagIdent tool (http://web.expasy.org/tagident/).