Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells

Abstract

Mass spectrometry (MS)-based proteomics typically employs multistep sample-preparation workflows that are subject to sample contamination and loss. We report an in-StageTip method for performing sample processing, from cell lysis through elution of purified peptides, in a single, enclosed volume. This robust and scalable method largely eliminates contamination or loss. Peptides can be eluted in several fractions or in one step for single-run proteome analysis. In one day, we obtained the largest proteome coverage to date for budding and fission yeast, and found that protein copy numbers in these cells were highly correlated (R(2) = 0.78). Applying the in-StageTip method to quadruplicate measurements of a human cell line, we obtained copy-number estimates for 9,667 human proteins and observed excellent quantitative reproducibility between replicates (R(2) = 0.97). The in-StageTip method is straightforward and generally applicable in biological or clinical applications.

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

“…These values are in good agreement with estimates reported in previous large‐scale proteomics studies (Nagaraj et al , 2011; Kulak et al , 2014; Hein et al , 2015), and this adds confidence to our use of γ‐tubulin data for calibration purposes. Also, we emphasize that although this calibration is critical for calculations of exact absolute copy numbers, any future correction of γ‐tubulin abundance would not affect relative numbers or any of the major conclusions.…”

“…Comprehensive studies on the proteomes of cells, tissues, or organisms have been reported (e.g. Addona et al , 2009; Nilsson et al , 2010; Beck et al , 2011; Nagaraj et al , 2011; for review, see Bensimon et al , 2012; Kulak et al , 2014; Wilhelm et al , 2014; Hein et al , 2015; Richards et al , 2015), but accurate quantitative information on proteins expressed at low levels remains scarce. As a consequence, published estimates for the abundance of specific proteins sometimes vary over several orders of magnitude.…”

Biallelic Mutations in MRPS34 Lead to Instability of the Small Mitoribosomal Subunit and Leigh Syndrome

“…Peptides reconstituted in 0.5% FA were loaded onto small cation exchange (Empore Cation Exchange-SR, Supelco Analytical) stage-tips made in-house. 43 Tips were washed with 20% acetonitrile (ACN), 0.5% FA and eluted over five fractions of increasing amounts (45À300 mM) freshly prepared ammonium acetate, 20% ACN, 0.5% FA, followed by a final elution of 5% ammonium hydroxide, 80% ACN. Fractions were concentrated by vacuum centrifugation and desalted on SDB-XC poly(styrene-divinyl-benzene; Supelco Analytical) stagetips as previously described.…”

“…Fractions were concentrated by vacuum centrifugation and desalted on SDB-XC poly(styrene-divinyl-benzene; Supelco Analytical) stagetips as previously described. 40,43 Peptides were reconstituted in 0.1% trifluoroacetic acid (TFA) and 2% ACN and analyzed by online nano-HPLC/electrospray ionization-MS/MS on a Q Exactive Plus connected to an Ultimate 3000 HPLC (Thermo-Fisher Scientific). Peptides were loaded onto a trap column (Acclaim C 18 PepMap nano Trap 3 2 cm, 100 mm I.D, 5 mm particle size, and 300 Å pore size; ThermoFisher Scientific) at 15 mL/min for 3 min before switching the pre-column in line with the analytical column (Acclaim RSLC C 18 PepMap Acclaim RSLC nanocolumn 75 mm 3 50 cm, PepMap100 C 18 , 3 mm particle size 100 Å pore size; ThermoFisher Scientific).…”

Nidogen-1 is a novel extracellular ligand for the NKp44 activating receptor

“…Next, for subsequent proteomic analysis, cells were processed by in-StageTip (iST) method. 74 Briefly, the pellets were lysed, reduced, alkylated in a single step using a buffer containing 2% (w/v) SDC (sodium deoxycholate), 10 mM TCEP (Tris(2-carboxyethyl)phosphine hydrochloride), 40 mM CAA (chloroacetamide), 100 mM Tris HCl pH 8.0, and loaded into StageTip. The lysates were diluted with 25 mM Tris pH 8.5 containing 1 µg of trypsin.…”