Trypsin Digestion Report

Quantification and trypsin digestion of polypeptides

Protein concentration was estimated by Bradford assay, and 100µg of total protein from each sample was subjected to in-solution trypsin digestion to generate peptides. Initially, treating the sample with 5µl of 100mM dithiothreitol in 50 mM ammonium bicarbonate for 30 min at 60ºC and alkylation with 200mM iodoacetamide in 50 mM ammonium bicarbonate at room temperature for 30 minutes reduced the protein disulphide bonds. Proteins were then digested with 4µg of sequencing grade-modified trypsin (Sigma) in 50 mM ammonium bicarbonate by incubating overnight at 37ºC. The trypsin digestion reaction was stopped by 1µl of 100% formic acid. The digested peptide solutions were centrifuged at 14000

The following parameters were used: nano-ESI capillary voltage, 3.3 KV; sample cone, 35 V; extraction cone, 4 V; transfer CE, 4 V; trap gas flow, (2 ml/minute); IMS gas (N2) flow, (90 ml/minute). To perform the mobility separation, the IMS T-Wave™ pulse height was set to 40 V during transmission and the IMS T-Wave™ velocity was set to 800 m/s. The traveling wave height was ramped over 100% of the IMS cycle between 8 and 20 V. The time of flight analyzer (TOF) was calibrated with a solution of 500 fmole/μl of human [Glu1]-Fibrinopeptide B (Sigma-Aldrich), and the lock mass acquisition was performed every 30 s by the same peptide delivered through the reference sprayer of the nano-LockSpray source at a flow rate of 500 nl/minute. This calibration set the analyzer to detect ions in the range of 50–2000 m/z.The mass spectrometer was operated in the “resolution mode” with a resolving power of 18,000 FWHM, and the data acquisition was performed in “continuum” format. The data was acquired by rapidly alternating between two functions: Function-1 (low energy) and Function-2 (high energy). In Function-1, only low energy mass spectra (MS) were acquired and in Function-2, mass spectra at elevated collision energy (MSE) with ion mobility were acquired. In Function-1, collision energy was set to 4 V in the trap region and 2 V in the transfer region. In Function-2, collision energy was set to 4 V in the trap region and is ramped from 20 V to 45 V in the transfer region. Each spectrum was acquired for 0.9 s with an interscan delay of 0.024