2 chloroacetamide caa

The following reagents were used in the experiments: dithiobis(succinimidyl propionate) (DSP) (Pierce, Waltham, MA, USA), 4-benzoylbenzoic acid N-succinimidyl ester (SuccBB) (Sigma, St. Louis, MO, USA), 3-aminopropyltriethoxysilane (APTES) (Acros Organics, Fair Lawn, NJ, USA), dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO, USA), Emulgen 913 (Kao Atlas, Osaka, Japan), triethylammonium bicarbonate (TEAB) buffer (1 M, for HPLC; Sigma-Aldrich, St. Louis, MO, USA), 100% acetonitrile for HPLC (CAN) (Merck, Darmstadt, Germany), 2,5-dihydroxybenzoic acid (DHB) matrix (Bruker Daltonics, Bremen, Germany), 2-chloroacetamide (CAA; Sigma-Aldrich, Darmstadt, Germany), trifluoroacetic acid (TFA) and chloroacetamide (Sigma, St. Louis, MO, USA), and 0.5 M Tris(2-carboxyethyl) phosphine hydrochloride (Bond-Breaker TCEP solution, neutral pH) (Thermo Scientific, Waltham, MA, USA).
PBSD buffer was prepared by dissolving a salt mixture, commercially available from Pierce (Waltham, MA, USA), in ultrapure water. All solutions used in this study were prepared using deionized ultrapure water (resistivity, 18.2 MΩ × cm) obtained with a Simplicity UV system (Millipore, Molsheim, France).

Label-free quantitative proteomic analysis was carried out as previously described [34 (link),35 (link)]. Briefly, HeLa cells (1 × 10⁶) were harvested, washed twice in PBS, and lysed in buffer containing 6M GnHCl, 20 mM tris(2-carboxyethyl)phosphine (TCEP; Pierce^TM, Thermo Fisher Scientific, Waltham, MA, USA), 40 mM 2-chloroacetamide (CAA; Sigma-Aldrich) in 100 mM Tris, pH 8.0. The lysate was heated to 95 °C for 2 min and then sonicated in a Bioruptor sonicator (Diagenode) at the maximum power setting for 10 cycles of 30 s each. The entire process of heating and sonication was repeated once, and then the sample was diluted 10-fold with digestion buffer (25 mM Tris, pH 8, with 10% acetonitrile). Protein extracts were digested for 4 h with endoproteinase lysC, followed by the addition of trypsin for overnight digestion. After digestion, peptides were purified and loaded for mass spectrometric analysis. Raw data were processed using the MaxQuant computational platform [46 (link)]. The peak list was searched against Human Uniprot databases, with an initial precursor and fragment tolerance of 4.5 ppm. The match between the run feature was enabled, and proteins were quantified across samples using the label-free quantification algorithm in MaxQuant as label-free quantification (LFQ) intensities [47 (link)].

The Sample Preparation by Easy Extraction and Digestion (SPEED) protocol was performed as described by Doellinger et al. [57 (link)]. Briefly, 100 mg of ground, frozen kidney cortex was dissolved in trifluoroacetic acid (TFA; Sigma-Aldrich, St. Louis, MO, USA) at a 1:4 v/v sample:TFA ratio. Reduction was performed using Tris(-carboxyethyl)phosphine (TCEP; Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 10 mM, and alkylation was simultaneously performed using 2-chloroacetamide (CAA; Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 40 mM. Protein concentration was determined using turbidity as described by Doellinger et al., and an aliquot of 150 µg of protein was adjusted to 0.75 µg/µL. Digestion was performed using a protein:trypsin ratio of 25:1 (sequencing-grade modified trypsin, PRV5111, Promega Fitchburg, WI, USA) at 37 °C for 20 h. Peptides were desalted using reverse-phase Vydac C18 Silica 96-well MACROspin plates (SNS SS18V-L, The Nest Group Inc., Ipswich, MA, USA), as detailed in the Supplementary Methods.