Dataset Information

Reduction-Free Thermolysin Digestion Enables Absolute Protein Quantitation in Plasma in 35 min from Sample to Result

ABSTRACT: We previously reported that the broad-specificity protease thermolysin yields reproducible, near-complete proteome digests within 1–2 min. Here we demonstrate rapid absolute protein quantitation in human plasma by combining reduction/alkylation-free thermolysin digestion on S-Trap cartridges with full-length stable isotope-labeled (SIL) protein standards and parallel reaction monitoring (PRM). Post-digest dilution of S-Trap eluates enabled immediate EvoTip loading, yielding LC-MS-ready samples in under 20 min. Using the anti-CD20 monoclonal antibody rituximab as a showcase, a lower limit of quantitation of 5 amol on-column per ~250 ng plasma protein injection could be achieved on an Orbitrap Exploris 480 in 60/100 samples-per-day mode. We applied the assay to 23 total pretreatment and on-treatment plasma samples from 12 chronic lymphocytic leukemia patients, demonstrating fit-for-purpose absolute quantitation in a clinical matrix. In 100 samples-per-day (SPD) mode, the full workflow enables on-demand absolute protein quantitation in 35 min from sample to fully acquired LC-MS data. The achieved sensitivity indicates the feasibility of transfer to other targets or matrices with lower concentrations. Because the required SIL proteins are used at femtomole levels (here, 40 fmol SIL rituximab spiked into 10 µg plasma protein), a single 10-µg SIL IgG vial is sufficient as internal standard for ~1,700 injections.

ORGANISM(S): Homo Sapiens

SUBMITTER: Rene Zahedi

PROVIDER: PXD077823 | panorama | Mon May 11 00:00:00 GMT+01:00 2026

REPOSITORIES: PanoramaPublic

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Reduction-Free Thermolysin Digestion Enables Absolute Protein Quantitation in Plasma in 35 min from Sample to Result.

Jiang Xuehui X Lao Ying Y Johnston James B JB Banerji Versha V Zahedi René P RP

Journal of proteome research 20260511 7

We previously reported that the broad-specificity protease thermolysin yields reproducible, near-complete proteome digests within 1-2 min. Here, we demonstrate rapid absolute protein quantitation in human plasma by combining reduction/alkylation-free thermolysin digestion on S-Trap cartridges with full-length stable isotope-labeled (SIL) protein standards and parallel reaction monitoring (PRM). Post-digest dilution of S-Trap eluates enabled immediate EvoTip loading, yielding LC-MS-ready samples ...[more]

PMID: 42113895

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Project description:We applied a non targeted mass spectrometric assay (LCMSE), to quantify 55 abundant plasma proteins in specimens from 31 healthy volunteers. Quantitation was done by HI3 peptide measurement using a single internal standard to estimate protein concentrations. Results for 7 apolipoproteins were compared with those obtained using isotope labelled standards, while 12 proteins were compared to routine immunoassays. Blood samples were obtained from 31 healthy volunteers (17 males; 14 females) with a median age of 46 years and a range of 22-67 years. Total plasma protein concentration was assayed with a BCA-assay (20) according to the manufacturer’s protocol (Thermo). Samples were diluted tenfold in 0.1% Rapigest SF (Waters Corporation, Milford, MA), 50 mM ammonium bicarbonate and heated at 95° C for 15 min. Subsequently, plasma samples were reduced with 5 mM dithiothreitol (60° C, 30 min) and alkylated with 15 mM iodoacetamide (ambient temperature, dark, 30 min). Proteolytic digestion was performed with modified trypsin (gold grade, Promega, Madison WI) at 0.3 units/µg protein, (37° C, 20 hours) unless indicated otherwise. Following digestion, Rapigest SF was broken down by adding 1% trifluoroacetic acid (pH<2, 37 °C, 45 min). Peptide solutions were centrifuged (20,000 x g, 10 min) and supernatant was collected. Prior to analyses a MASSPREP protein digestion standard (Waters Corporation, ADH1 or ENO from Saccharomyces cerevisiae) was added for quantitation purposes. LC-MS analyses were performed using ~ 0.21 µg of the final plasma protein digest mixtures (384 times total dilution) unless indicated otherwise. LC separations of tryptic peptides were performed with a NanoAcquity system (Waters Corporation), coupled to a Synapt G2 quadrupole time of flight mass spectrometer (Waters Corporation, Manchester, UK). Accurate mass precursor and fragment ion LC-MS data were collected in data independent LCMSE mode of acquisition. Continuum LC-MS data were processed using ProteinLynx GlobalSERVER version 2.5 (PLGS 2.5, Waters Corporation). Parameter settings: digest reagent trypsin, allow 1 ‘missed cleavage’, search tolerances automatic, typically 5 ppm for precursor and 15 ppm for product ions, fixed modification cysteine carbamidomethylation, and variable modification methionine oxidation. Protein identifications were obtained searching the human SwissProt entries of an UniProt database (release 13.2). This database was modified to include N-terminal processing of proteins using protein maturation device software, with ADH1 and ENO1 of S. cerevisiae appended as internal standard to address technical variation and allow concentration determinations. Estimation of false-positive identification rates was done by searching a randomized version of the abovementioned human protein database generated within PLGS 2.5. Data were exported as csv-files for further, detailed analysis. Stringent criteria were applied for quantitation, protein identifications were only considered significant if reported in 14 or more samples. Protein false positive identification rates were estimated using the criteria mentioned above and no false positives were identified in these searches. DATASET: KC1-31: 31 healthy volunteers QC1-10: 10 times injection of a single sample to ascertain analytical variation over 9 days of measurements. 20120802_QC1-QC6: 6 pooled plasma samples worked up and analysed during a single day of measurements to ascertain Intra-assay variation. 20120427_S2,S4;20120525_S18,S19;20120626_S3,S4;20120802_QC7: 7 pooled plasma samples processed and analysed during 3 months of routine measurements to analyse Inter-assay variation 20130416_s1-s5: pooled plasma samples spiked with a QCONCATAMER for 11 apolipoproteins labelled by heavy Lysine and Arginine (+6.02 AMU) to quantify apolipoproteins by internal isotope labelled standard.

Project description:Background: Diclofenac is a non-steroidal anti-inflammatory drug frequently found in the aquatic environment. Previous studies have reported histological changes in the liver, kidney and gills of fish at concentrations similar to those measured in treated sewage effluents (~1 µg/L). Analyses or predictions of blood plasma levels in fish allow a direct comparison with human therapeutic plasma levels, and may therefore be used to indicate a risk for pharmacological effects in fish. To relate internal exposure to a pharmacological interaction we therefore investigated global hepatic gene expression together with bioconcentration to blood plasma and liver of rainbow trout (Oncorhynchus mykiss) exposed to waterborne diclofenac. Results: At the highest exposure (81.5µg/L) the fish plasma concentration reached ~88% of the human therapeutic levels (Cmax) after two weeks. Using an oligonucleotide microarray followed by quantitative PCR we found extensive effects on hepatic gene expression at this concentration, and some genes were regulated down to the lowest concentration tested (1.6 µg/L) corresponding to ~1.5% of the human Cmax. Functional analysis of differentially expressed genes revealed effects on biological processes such as inflammation and immune response, in agreement with the expected mode of action of diclofenac. In contrast to some previously reported results, the bioconcentration factor was found to be stable (4.02±0.75 for blood plasma and 2.54±0.36 for liver) regardless of the water concentration. Conclusions: Hepatic gene expression is affected in fish at blood levels similar to and below the human Cmax. This adds confidence to the use of fish blood plasma levels to predict risks for pharmacological responses in fish. The microarray results indicate similarities in the mode of action of diclofenac between humans and fish. The identified set of regulated genes may contain useful exposure biomarkers to be used in complex exposure scenarios. Juvenile rainbow trout was exposed to waterborne diclofenac at a series of different concentrations for 14 days. A flow-through system was used for eight aquaria (two per concentration), each containing 12 trouts. Nominal exposure concentrations: 1µg/L, 10 µg/L, 100 µg/L and control. Total RNA for biotinylated aRNA synthesis was extracted from the liver. Samples of aRNA from two fish from the same aquarium were pooled before the assay. In total, four biochips were analyzed corresponding to 32 microarrays. As we used two aquaria per concentration, these 32 microarrays allowed the analyses of four aRNA pools (eight fish) per aquarium, which is equal to eight pools per concentration of diclofenac.

Project description:In order to identify molecular targets of USP2-45 in the small intestine, we carried out a quantitative proteomics iTraq (Isobaric tag for relative and absolute quantitation) screen to compare proteins from WT and knockout mice. With this analysis we identified, among others, the scaffolding protein Na(+)/H(+) Exchange Regulatory cofactor NHERF4 to be up-regulated (4.81-fold) in the duodenum of Usp2-KO mice. Methods : Usp2-KO and WT littermates males were entrained to 12h L:12h D for at least two weeks in light-light housing boxes. Mice were sacrificed by cervical disolcation at ZT13 under red dim light and duodenal mucosa was dissected by scraping. The tissue samples were quickly frozen in liquid nitrogen before total protein extraction. Proteins were extracted in a lysis buffer containing 8M Urea, 20mM Tris-HCl, pH 7.5, 14 µg/ml Aprotinin, 0.7 µg /ml PepstatinA 0.7 µg /ml Leupeptin, 1 mM NaVO4, 10 mM Na-Pyrophosphate, 50 µM MG132 and 1mM PMSF. Protein concentration was measured by Bradford quantitation. Relative quantitation of proteins in 4 pools of 3 KO and 4 pools of 3 WT tissue samples was carried out by iTRAQ (Isobaric Tags for Relative and Absolute Quantitation). After reduction and alkylation of cysteines, proteins were precipitated, redissolved in 8M Urea buffer and digested with trypsin (1:50 w:w) overnight. 80ug of digested material for each sample was labeled by reaction with one vial of iTRAQ 8-plex reagent (ABSciex). Samples were pooled , desalted and fractionated by off-gel electrofocusing. The 24 fractions obtained were analysed by nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) on a hybrid linear trap LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) on a two-hour gradient. Full MS survey scans were performed at 60’000 resolution and the ten most intense multiply charged precursor ions detected in the full MS survey scan were selected for HCD (Higher energy Collision Dissociation) fragmentation and Orbitrap analysis (7500 resolution). Each spectrum was acquired at two relative collision energies (35% and 45%) and the spectra were summed. Data files were analysed with MaxQuant 1.3.0.5 incorporating the Andromeda search engine. Further data analysis was performed using the R statistical programming language version 2.15.2 (R core team 2012). Intensities for the reporter ions 113 to 121 were normalized using the Variance Stabilizing method (R package version 3.26.0). Proteins with non-zero intensities in all channels were evaluated for differential expression using the Local-Pooled-Error method (R package version 1.32.0) followed by correction for multiple testing according to Benjamini and Hochberg.

Dataset Information

Reduction-Free Thermolysin Digestion Enables Absolute Protein Quantitation in Plasma in 35 min from Sample to Result

Publications

Reduction-Free Thermolysin Digestion Enables Absolute Protein Quantitation in Plasma in 35 min from Sample to Result.

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