Sequence coverage by multiple reads in shotgun proteomics to validate single amino acid variants
Protocol
Sample preparation
The samples of the human embryonic kidney cell line HEK 293 were analyzed in three biological replicates. Cell pellets of one million cells each were resuspended in lysis buffer containing 4% SDS in 100 mM TEABС, pH 8.5, incubated for 5 minutes and subjected to sonication by Qsonica Q55 ultrasonic homogenizer (Qsonica, Newtown, CT, USA) at 70% amplitude using 10 series of 10 one-second-duration impulses. After that, the samples were incubated for 10 minutes at 85°C and centrifuge for 10 minutes at 16000 × g. Later on, the disulfide bonds of the proteins were reduced by the addition of dithiothreitol (DTT) up to 5 mM and incubation for 30 minutes at 56°C and the cysteines were alkylated with chloroacetamide (CAM) in the final concentration of 10 mM for 15 minutes at the room temperature in the darkness. 
At the next step, 30 μg of total protein was taken from each sample and 4 volumes of cold (-20°C) acetone were added to the samples. The samples were vortexed and left for 120 minutes at -20°C to let the proteins thoroughly precipitate. After the incubation period, the samples were centrifuged for 10 minutes at 13000-15000 × g at +4°C followed by rinsing with cold acetone twice without mixing. Then the acetone was carefully removed and the tubes were left with the lids open for 30 minutes to let the pellets dry up. 
For the digestion, 30 μl of each protease namely trypsin (Promega Gold), LysC and GluC (all from Promega, USA) in the in 50 mM TEABC were added straight to the pellets. The final proportion of a protease to the protein was 1:50. Then the samples were incubated overnight at +37°C. To stop the reaction and let the SDC precipitate, TFA up to 1% was added to each tube. 
For the peptide desalting and clean up, 
For the peptide desalting and cleanup the in-house made stage tips containing SDB-RPS membrane (Empore-3M, CDS Analytical, Oxford, PA, USA) were used. The tips were prepared with the use of 3 pieces of membrane in each tip. First, two volumes of ethyl acetate were added to the samples in order to dissolve the SDC precipitate and other unwanted contaminants. The samples were vortexed followed by quick centrifugation for 2 min at 6000 rpm (centrifuge BioSan Multi-spin MSV-6000, BioSan, Riga, Latvia). Then the upper phase was discarded and the procedure was repeated for three times. Next, the samples were loaded into the tips and the tips were centrifuged at 1200 rpm (centrifuge BioSan Multi-spin MSV-6000, BioSan, Riga, Latvia)(about 70 × g) until the solution has passed through the membrane. At the next step, 100 μl of 1% TFA covered by 50 μl of ethyl acetate were passed through the tips at the same speed in order to remove the remnants of SDC. After that, the washing with 100 μl 0.2% TFA was performed at the same speed. The peptides were eluted by passing 60 μl of 70% acetonitrile (CAN) with 5% NH4OH through the tips into the clean tubes at the speed as low as 1000 rpm (about 50 × g) in the same centrifuge. The peptide samples were dried up in the vacuum concentrator (Labconco, Kansas City, MO, USA). 
Liquid chromatography and mass spectrometry. 
For the LC-MS analysis, the samples were reconstituted in 0.1% TFA and loaded onto an Acclaim PepMap 100 C18 (100 μm × 2 cm) trap column in the loading mobile phase (2% ACN, 98% H2O, 0.1% TFA) at 10 μl/min flow and separated at 40°C on a 500 mm 75 μm inner diameter Thermo ScientificTM Acclaim™ PepMap™ 100 C18 LC column with particle size 2 μm. Reverse-phase chromatography was performed with an Ultimate 3000 Nano LC System (Thermo Fisher Scientific), which was coupled to the Orbitrap QExactive HF mass spectrometer (Thermo Fisher Scientific) via a nanoelectrospray source (Thermo Fisher Scientific). Water containing 0.1% (v/v) formic acid (FA) was used as mobile phase A and ACN containing 0.1% FA (v/v), 20% (v/v) H2O as mobile phase B. Peptides were eluted from the trap column with a linear gradient: 3–35% solution B (0.1% (v/v) formic acid, 80% (v/v) acetonitrile) for 105 min; 35-55% B for 18 min, 55-99% B for 0.1 min, 99% B during 10 min, 99-2% B for 0.1 min at a flow rate of 300 nl/min. After each gradient, the column was reequilibrated with A for 10 min. MS data was collected in DDA mode (TopN=15). MS1 parameters were as follows: 120K resolution, 350-1400 scan range, max injection time – 50 msec, AGC target – 3x106. Ions were isolated with 1.2 m/z window, preferred peptide match and isotope exclusion. Dynamic exclusion was set to 30 s. MS2 fragmentation was carried out at 15K resolution with HCD collision energy 28, max injection time – 80 msec, AGC target – 1x105. Other settings: charge exclusion - unassigned, 1, 6-8, >8. 
Data analysis
The raw files obtained from the instrument were converted into the mzML format using ThermoRawFileParser. The spectra were searched against the SwissProt human proteomic data base downloaded in March of 2021 and containing 20497 entries. The proteomic search and protein identification were performed using IdentiPy search engine v.0.3.3.16 followed by the post-search treatment and result filtration by Scavager v.0.2.4. For the search a corresponding protease was chosen as a parameter. For trypsin and Lys-C the number of allowed missed cleavages was 2, while for Glu-C this number was 3. Mass accuracy for the precursor and the fragment ions were 10 ppm and 0.01 Da respectfully. Carbamidomethylation of Cys was accounted as a fixed modification and oxidation of Met accounted as a variable modification. 
The PSMs, identified peptides and proteins were filtered with Scavager according to the target-decoy strategy with 1% false-discovery rate cut-off.