Precipitated proteins (1 μg) from ST-LGP2/protein and ST-CH/protein complexes were used in each nano–LC-MS/MS experiment. Proteins were digested overnight at 37°C with 20 μl of a trypsin solution prepared as follows: high-performance LC (HPLC) water, trypsin-sequencing grade (12.5 μg/ml; #PRV5111, Promega), 10% HPLC grade acetonitrile (ACN), and 25 mM ammonium carbonate (#09830, Sigma Aldrich). The resulting peptide extracts were speed-vac–dried and dissolved in 12 μl of HPLC grade water and 0.1% formic acid. Samples (5 μl) were then used for nano–LC-MS/MS analysis using a nanochromatography system (Easy nLC, Proxeon) connected online to a LTQ Velos Orbitrap (Thermo Fisher Scientific) mass spectrometer. A 2-cm-long and 5-μm particle size C18 Easy column (Proxeon) was used for peptide trapping and desalting. A 10-cm-long and 3-μm particle size C18 Easy column (Proxeon) was used for peptide separation. The peptide elution gradient was from 100% buffer A (HPLC grade water and 0.1% formic acid) to 35% buffer B (HPLC grade ACN and 0.1% formic acid) in 60 min using a constant flow of 300 nl/min. MS spectra were acquired on the Orbitrap analyzer at resolution R = 30,000. After each MS spectrum, an automatic selection of the 20 most intense precursor ions was activated with a 15-s dynamic exclusion delay to acquire MS/MS spectra on the LTQ Velos analyzer using CID fragmentation mode at 35% relative resonant activation energy for 40 msec. Raw data were preprocessed using ProteomeDiscoverer version 1.4 (Thermo Fisher Scientific). Preprocessing consisted of MS/MS spectrum averaging of spectra from equal precursor molecular weight, with a 5 parts per million (ppm) mass tolerance and a 90-s time window. No threshold was applied either to MS ions intensities or to MS/MS fragment ion intensities. All spectra were analyzed using Mascot version 2.3 (Matrix Science). Queries were performed against a nonredundant database of 20,352 human protein sequences from Swiss-Prot (release 2013-02). Mascot was run in MS/MS Ion search mode with the following parameter settings: no fixed modification, variable modification [oxidation (Met), phosphorylation (Ser, Thr, and Tyr), acetylation (Lys and N-term), deamidation (Asn and Gln)], precursor mass tolerance of 7 ppm, fragment ions mass tolerance of 0.5 Da, two missed cleavages, and trypsin as the digestion enzyme. False discovery rates were calculated for each peptide by the percolator node for validation of peptide identification. To reduce the number of nonspecific hits, if the protein hit presented a score of >20 in any of the three ST-CH replicates, the protein was immediately excluded from the list. A second filter was added whereby the protein had to have a score of >60 for two of the three replicates and could have a score of >40 for the third replicate (fig. S1A).

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.