PXD000347

PXD000347 is an original dataset announced via ProteomeXchange.

Title	Quantitation of plasma protein concentrations by mass spectrometry without the use of isotopic standards. C4PR_LIV
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.
HostingRepository	PRIDE
AnnounceDate	2017-10-24
AnnouncementXML	Submission_2017-10-24_06:08:08.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Gertjan Kramer
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	6x(13)C labeled residue; monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Synapt G2 MS

Revision	Datetime	Status	ChangeLog Entry
0	2013-07-17 06:14:14	ID requested
1	2015-10-20 05:00:20	announced
2	2015-10-21 03:36:42	announced	Updated publication details.
3	2015-10-24 09:50:00	announced	Updated publication details.
4	2017-02-16 00:30:30	announced	Updated project metadata.
5	2017-10-24 03:00:32	announced	Updated project metadata.
⏵ 6	2017-10-24 06:08:09	announced	Updated project metadata.

Kramer G, Woolerton Y, van Straalen JP, Vissers JP, Dekker N, Langridge JI, Beynon RJ, Speijer D, Sturk A, Aerts JM, Accuracy and Reproducibility in Quantification of Plasma Protein Concentrations by Mass Spectrometry without the Use of Isotopic Standards. PLoS One, 10(10):e0140097(2015) [pubmed]

curator keyword: Biomedical

submitter keyword: Mass Spectrometry, quantitation, Plasma proteins, Immunoassay, Proteomics, LCMSE

Gertjan Kramer
contact affiliation	Proteomics Unit
contact email	gertjan.kramer@embl.de
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD000347
     1. Label: PRIDE project
     2. Name: Quantitation of plasma protein concentrations by mass spectrometry without the use of isotopic standards. C4PR_LIV