Table listing the lowest detection level within the linear response range for one peptide per protein in the 11-plex AFFIRM assay in the three different magnetic bead systems.

<p>The assay for each target protein in the three systems is rated into three grades of assays, A-C. Rating is based on lowest detected concentration in the linear response range where linear response is defined by an R² value above 0.9. The assay grades are defined as; A = 0.05–1 ng/ml; B = 5–12.5 ng/ml; C = 25+ ng/ml of lowest detected peptide level in the linear response range. This detection level is combined with the R² value where the assay grade is downgraded one level (i.e. from A to B or from B to C) if the R² value is below 0.9. The lowest concentration of detected target proteins within the linear response is displayed as both ng/ml and fmol/ml for respective bead system. The epoxy system could only detect 6 out of the 11 proteins.</p

Schematic figure and features describing the three different scFv magnetic bead coupling strategies; epoxy, streptavidin and anti-FLAG.

<p>The epoxy format is different to the other two in that scFv are coupled directly to the magnetic beads through a covalent coupling to any lysine residue on the scFv. This results in randomly oriented (non-oriented) scFv on the beads. As opposed to the streptavidin-coated beads and M2 anti-FLAG beads where the scFv is immobilized through coupling to the bead surface through a linker at the back-end of the scFv and thus an oriented coupling that is not interfering with the binding site of the scFv. The steptavidin and anti-FLAG formats also require much less scFv and shorter coupling time.</p

Single-plex AFFIRM captures of target proteins KER19 and P85A performed on-bead (left) according to the standard protocol and in-solution (right) using the streptavidin system.

<p>The in-solution captures are allowing antibody and antigen to bind before adding magnetic beads for isolation. Target protein was spiked in serum background from 125 down to 1 ng/ml concentration. Two scFv were used for capture of each protein.</p

Linear response range for one peptide per target protein in the11-plex AFFIRM experiment in a) the anti-FLAG format and b) the streptavidin format, demonstrating detection with linear response down to low ng/ml concentrations for the majority out of the target proteins.

<p>Linear response range for one peptide per target protein in the11-plex AFFIRM experiment in a) the anti-FLAG format and b) the streptavidin format, demonstrating detection with linear response down to low ng/ml concentrations for the majority out of the target proteins.</p

Single-plex captures of target proteins IL6 and GAK in the streptavidin (a, b) anti-FLAG (c, d) and epoxy (e, f) scFv—Magnetic bead coupling system.

<p>Target proteins were enriched for from serum background at spike levels of 50, 5, 0.5 ng/ml and 50 pg/ml. Each concentration point was run in triplicates.</p

Multiplexed (11-plex) captures of target proteins IL6 and GAK using the streptavidin (a, b) anti-FLAG (c, d) and epoxy (e, f) scFv—Magnetic bead coupling system.

<p>Target proteins were enriched for from serum background at spike levels of 100, 50, 25, 12.5, 5, 1, 0.5, 0.1 ng/ml and 50 pg/ml. Each concentration point was run in triplicates.</p

AFFIRMA Multiplexed Immunoaffinity Platform That Combines Recombinant Antibody Fragments and LC-SRM Analysis

Targeted measurements of low abundance proteins in complex mixtures are in high demand in many areas, not the least in clinical applications measuring biomarkers. We here present the novel platform AFFIRM (AFFInity sRM) that utilizes the power of antibody fragments (scFv) to efficiently enrich for target proteins from a complex background and the exquisite specificity of SRM-MS based detection. To demonstrate the ability of AFFIRM, three target proteins of interest were measured in a serum background in single-plexed and multiplexed experiments in a concentration range of 5–1000 ng/mL. Linear responses were demonstrated down to low ng/mL concentrations with high reproducibility. The platform allows for high throughput measurements in 96-well format, and all steps are amendable to automation and scale-up. We believe the use of recombinant antibody technology in combination with SRM MS analysis provides a powerful way to reach sensitivity, specificity, and reproducibility as well as the opportunity to build resources for fast on-demand implementation of novel assays

Additional file 1: Table S1. of Proteomic analysis of breast tumors confirms the mRNA intrinsic molecular subtypes using different classifiers: a large-scale analysis of fresh frozen tissue samples

The clinical parameters of the tumors used in the 2D-DIGE analysis are given in this table: Sex, tumor type, BRCA type, age at diagnosis, ER status, PgR status, RNA subtype classification according to Hu, SĂśrlie, and PAM50 and the associated gel number. (PDF 115 kb

Additional file 3: Table S3. of Proteomic analysis of breast tumors confirms the mRNA intrinsic molecular subtypes using different classifiers: a large-scale analysis of fresh frozen tissue samples

The table lists all the proteins identified using MaxQuant, the Protein IDs, protein description, posterior error probability, and confidence score. (PDF 2732 kb

Automated Selected Reaction Monitoring Software for Accurate Label-Free Protein Quantification

Selected reaction monitoring (SRM) is a mass spectrometry method with documented ability to quantify proteins accurately and reproducibly using labeled reference peptides. However, the use of labeled reference peptides becomes impractical if large numbers of peptides are targeted and when high flexibility is desired when selecting peptides. We have developed a label-free quantitative SRM workflow that relies on a new automated algorithm, Anubis, for accurate peak detection. Anubis efficiently removes interfering signals from contaminating peptides to estimate the true signal of the targeted peptides. We evaluated the algorithm on a published multisite data set and achieved results in line with manual data analysis. In complex peptide mixtures from whole proteome digests of <i>Streptococcus pyogenes</i> we achieved a technical variability across the entire proteome abundance range of 6.5–19.2%, which was considerably below the total variation across biological samples. Our results show that the label-free SRM workflow with automated data analysis is feasible for large-scale biological studies, opening up new possibilities for quantitative proteomics and systems biology