PPP reference specimens

The primary specimens were sets of four reference specimens prepared under the direction of the HUPO PPP Specimens Committee by BD Diagnostics for each of three ethnic groups Caucasian-American (B1), African-American (B2), and Asian-American (B3). Each pool consisted of 400 mL of blood each from one male and one post-menopausal female healthy, fasting donor, collected into 10 mL tubes in a prescribed sequence (see Supplementary Protocol) after informed consent. Very large pools were rejected as...

Antibodybased measurements of the HUPO reference specimens

The PPP reference specimens were distributed to four different laboratories for immunoassay or antibody microarray analysis. Each of the four sites used a distinct technology for analyzing the specimens. The 39 immunoassays performed on DB clinical analyzers were based on immunonephelometric methods (33 tests) and sandwich-like enzyme immunoassays (6 tests) that use antibody-coated magnetic chromium dioxide particles. The GNF measured 88 different serum proteins using microarray-based sandwich...

Selditof analysis for protease inhibitor studies

Plasma samples from 20 individuals were collected into identical EDTA-contain-ing tubes with a patented mechanical separator, either with or without the presence of a protease inhibitor cocktail. After collection, samples were processed as quickly as possible (within 15 min) into plasma, hence time zero analyses were from aliquots that were frozen at this earliest time point. Analysis was performed using H50 chips, as per manufacturer's instructions. Briefly, the chips are preincubated with 50...

Consistent alterations in specific protein abundances

We then examined whether specific proteins, as opposed to all the proteins in general, were consistently highest or lowest in a certain preparation type. Evidence for such a bias would be indicated by multiple specimen sets showing agreement in the alteration of the concentration of a specific protein, e.g., if all three of the BD specimen sets showed a certain protein higher in a certain preparation method. We identified the proteins that always gave a highest value in one particular...

Annotation of the Hupo Ppp core datasets

From the inception, HUPO has intended that the Plasma Proteome Project facilitate extensive and innovative annotation of the human plasma and serum pro-teome. A large element of the Jamboree Workshop was focused on collaborative annotation. Several papers in this issue report on those collaborations. Ping et al. 56 emphasize use of peptide identification results from MS MS to reveal cleavage of signal peptides, proteolysis within hydrophobic stretches in transmembrane protein sites, and PTMs....

Choice of specimen and collection and handling variables

Pre-analytical variables can alter the analysis of blood-derived samples. Publications and protocols are generally deficient in this regard. Besides preparing the reference specimens of serum and plasma for direct comparisons, we undertook special studies on choice of sample type, stability during storage, use of protease inhibitors, and criteria for clinical standardization. The Specimens Committee concluded (Rai et al. 23 ) that plasma is preferable to serum, due to less degradation ex vivo...

DB immunoassays

DB immunoassays (see Supplemental Tab. 1, http www.vai.org vari labs haab. asp) were performed on a Behring Nephelometer (BN) II (2.2 D, serial no. 330135) and on a Dimension (DIM) RxL (serial no. 970933-AX) from DB (Deerfield, IL) with the HUPO PPP specimens 8 . Most tests performed are approved by the Food and Drug Administration (FDA) only for serum samples, as outlined in the manufacturer data sheets. Tests for ferritin (FERR), soluble transferrin receptor (sTfR), cardiac troponin I (cTNI),...

Linkage of MS data and antibodybased measurements

Another valuable use of these data for the PPP was to investigate relationships between the quantitative antibody-based measurements and the MS information derived from other work within the PPP. Based on the informatics integration methods (see Adamski et al., this issue), 9504unique IPI proteins were included in the combined data (see The link between the MS data and the antibody-based measurements was made through IPI numbers. Two different search methods were used to find IPI numbers that...

Analyses of human plasma and serum proteomes using HUPO filter criteria

All the data described above were analyzed using the XCorr DCn Sf criteria. From experience, we know that a substantial number ofthe proteins identified by a single peptide using these criteria are incorrect and the probability of a protein being correctly identified increases with the number of unique peptides identified. However, for biomarker discovery it is better to have a less stringent filter so that potentially interesting low-abundance proteins will not be excluded from the analysis....

Reference materials

Reference materials (RMs) are applied in a variety of analytical procedures to perform harmonized quantifications of analytes. Methods can be standardized by the use of common RMs 30 . The use of international RMs ensures world wide uniformity in most analytical measurements and provides users with reliable and comparable quantitative information based on operationally defined conventional true values in the sense of a gold standard of the certified analytes. The agreement of a measured average...

Systematic protein array pixelation of the human serum proteome

Protein array pixelation of the HUPO serum sample, BDCA02-Serum, was performed using a method similar to that used for the plasma sample, except this method incorporated several refinements to further improve coverage of the pro-teome (Fig. 5A). The major protein depletion was performed on 415 mL (35.3 mg) of serum using a dual MARS column. A total of 4.3 mg of unbound proteins were recovered, indicating that approximately 88 of the total serum protein content was removed in this sample...

HUPO reference sample collection protocol

HUPO reference samples were collected at, and using materials from BD Diagnostics. For each ethnic group (Caucasian, Asian American, African American), blood was acquired by venipuncture from one male donor and one female donor. Donors were tested and determined negative for HIV-1 and HIV-2 antibody, HIV-1 antigen (HIV-1), Hepatitis B surface antigen (HBsAg), Hepatitis B core antigen (anti-HBc), Hepatitis C virus (anti-HCV), HTLV-I II antibody (anti-HTLV-I II), and syphilis. Blood intended for...

Systematic protein array pixelation ofthe human plasma proteome

The profiling of human plasma proteome began with major protein depletion from a total of 193 mL (14.5 mg) plasma (BDCA02-Heparin) using the MARS antibody column (Fig. 3A). Following depletion, 2.4 mg of unbound proteins were recovered, indicating that the six targeted proteins constituted approximately 83 of plasma proteins in this sample. Analysis ofthe bound fraction by 2-DE showed that the bound proteins were the six targeted proteins with no apparent evidence of other proteins 9 . The...

Dr Eric W Deutsch

Institute for Systems Biology, 1441 N 34th Street, Seattle, Overview of the HUPO Plasma Proteome Project Results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of3020 proteins and a publicly-available database Gilbert S. Omenn, David J. States, Marcin Adamski, Thomas W. Blackwell, Rajasree Menon, Henning Hermjakob, Rolf Apweiler, Brian B. Haab, Richard J. Simpson, James S. Eddes, Eugene A. Kapp, Robert L. Moritz, Daniel W. Chan,...