Mass Spectrometry

Sample preparation was performed (Guttman et al., 2009) by diluting crude lysates of RAY and Deimos-Minion in TNE (50 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA) buffer and adding RapiGest SF reagent (Waters Corp.) to a final concentration of 0.1%. Samples were then boiled for 5 min followed by addition of 1 mM (final concentration) of TCEP [Tris (2-carboxyethyl) phosphine] and incubated at 37°C for 30 min. Afterward, carboxymethylation of samples was done with 0.5 mg/ml of iodoacetamide for 30 min at 37°C followed by neutralization with 2 mM TCEP (final concentration). Trypsin (trypsin: protein ratio – 1:50) was used overnight at 37°C to digest the crude lysates prepared as above. The samples were treated with 250 mM HCl at 37°C for 1 h followed by centrifugation at 14000 rpm for 30 min at 4°C to degrade and remove RapiGest. The soluble fraction was then added to a new tube and Aspire RP30 desalting columns (Thermo Fisher Scientific) were used for extraction and desalting of the peptides.

High pressure liquid chromatography (HPLC) coupled with tandem mass spectroscopy (LC-MS/MS) using nano spray ionization was used to analyze Trypsin-digested peptides (McCormack et al., 1997). A TripleT of 5600 hybrid mass spectrometer (ABSCIEX) interfaced with nano-scale reversed-phase HPLC (Tempo) using a 10 cm-100-micron ID glass capillary packed with 5-μm C18 Zorbax^TM beads (Agilent Technologies, Santa Clara, CA) was used to perform the nano-spray ionization experiments. By using a linear gradient (5–60%) of ACN (Acetonitrile) at a flow rate of 250 μl/min for 1 h, peptides were eluted from the C18 column into the mass spectrometer The ACN gradient was created using these buffers: buffer A (98% H₂O, 2% ACN, 0.2% formic acid, and 0.005% TFA) and buffer B (100% ACN, 0.2% formic acid, and 0.005% TFA). In a data-dependent manner MS/MS data were acquired in which the MS1 data was acquired for 250 ms at m/z of 400 to 1250 Da and the MS/MS data was acquired from m/z of 50 to 2,000 Da. For Independent data acquisition (IDA) parameters MS1-TOF 250 ms, followed by 50 MS2 events of 25 ms each. The IDA criteria; over 200 counts threshold, charge state of plus 2–4 with 4 s exclusion window. Finally, MASCOT^® (Matrix Sciences) was used to analyze the collected data and Protein Pilot 4.0 (ABSCIEX) was used for peptide identifications.