Validation of peptide epitope microarray experiments and extraction of quality data

Abstract. Within the last decade, the development of antigen microarray slides has enabled the simultaneous measurement of serum reactivity to hundreds of peptides in a single biological sample. Despite this considerable scientific progress, many issues remain regarding the quality, analysis and interpretation of the data these slides produce. There is currently no accepted approach to guide data analysis, and researchers use a wide variety of statistical methods and software tools. We designed and implemented a laboratory experiment to assess the reliability and range of measurement of peptide microarray data, and present graphical and statistical procedures for pre-processing so that quality data can be extracted for addressing biological hypotheses

Intersection between PAM and the Exclusive Recognition analysis.

<p>The table lists peptides that were commonly defined by PAM (peptides with significant differences in the intensity of the response vs. the reference group D−R−) and as well in the ‘exclusive recognition analysis’ (never above a threshold for detection in the D−R− group). Peptides marked with a star (*) were above this threshold in all patients in the respective group but never in serum from D−R− patients. The average Q-value is the absolute difference between PAM Q-score for D−R− and the Q-score for each respective group. Higher average Q-value indicates the probability that the peptide is differently recognized between the respective group and the D−R− (reference) group.</p><p>At 6 months, possible cross-reactivity to serum IgG for the epitope QPGENEVRPHAGVID (HCMV UL102) and the aminoacid sequence from a DNA packaging tegument protein UL17 from <i>Herpes Simplex virus-1</i>, which shows a matching alignment of 5 amino acids without any mismatch. Note at 12 months, a possible cross-reactivity of serum IgG for the epitope AQLDLEADPTAREGE (HCMV UL35) and the aminoacid sequence from a protein from <i>Epstein-Barr virus</i>, RNGATFSKGDIEGNF (HCMV US30), the <i>Human herpesvirus-6A</i> protein, the epitope YPAVTTVYPPSSTAK and YDDESWRPLSTVDDH directed against proteins from <i>Human herpesvirus 8</i>, could be found. At 24 months, no matches (using the criteria outlined in materials and methods) between serum CMV epitope recognition and proteomes of other human herpesviruses were found.</p

Epitopes predicted by the Exclusive RECOGNITION analysis.

<p>The peptides presented are only detectable in serum from at least 4 out of 5 individuals in the respective group (D−R+, D+R− or D+R+, 6, 12 and 24 months post-HSCT) but never in the group D−R− at 6, 12, and 24 months post-HSCT.</p

Result of the microarray significant profiles analysis (MaSigPro).

<p><b>a</b>) Venn diagram with the number of significant peptides obtained in the three comparisons (each patient group vs. D−R−. The lists of peptides provided in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089648#pone.0089648.s009" target="_blank">Table S5</a> represents the entire set of peptides contained in the Venn diagram. These peptides were also grouped into 9 clusters (default value) according to their recognition profile (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089648#pone.0089648.s009" target="_blank">Table S5</a>). <b>b–c</b>) Cluster analyses using CMV peptides that were differentially recognized in serum from patients, based on the D−/R− status. Three representative peptide clusters are reported, one for each analysis: D−R+ vs. D−R− (top), D+R− vs. D−R− (middle), D+R+ vs. D−R− (bottom). b) The consistency of the CMV epitope response in the cluster is visualized using the continuous peptide recognition profile across all the samples. Each peptide in the cluster is represented with a different color. c) The group-averaged CMV epitope recognition profiles (for different time points after HSCT) are shown to visualize differences (between the different patient groups) for CMV peptides selected in each cluster. Each group is represented with a different color (red = D−R−, green = D−R+, blue = D+R−, cyan = D+R+). Below the figures, peptides in the three clusters are listed. All the identified clusters and peptides are reported in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089648#pone.0089648.s008" target="_blank">Table S4</a> in greater detail.</p

Intersection between PAM and the Exclusive Recognition analysis.

<p>The table lists peptides that were commonly defined by PAM (peptides with significant differences in the intensity of the response vs. the reference group D−R−) and as well in the ‘exclusive recognition analysis’ (never above a threshold for detection in the D−R− group). Peptides marked with a star (*) were above this threshold in all patients in the respective group but never in serum from D−R− patients. The average Q-value is the absolute difference between PAM Q-score for D−R− and the Q-score for each respective group. Higher average Q-value indicates the probability that the peptide is differently recognized between the respective group and the D−R− (reference) group.</p><p>At 6 months, possible cross-reactivity to serum IgG for the epitope QPGENEVRPHAGVID (HCMV UL102) and the aminoacid sequence from a DNA packaging tegument protein UL17 from <i>Herpes Simplex virus-1</i>, which shows a matching alignment of 5 amino acids without any mismatch. Note at 12 months, a possible cross-reactivity of serum IgG for the epitope AQLDLEADPTAREGE (HCMV UL35) and the aminoacid sequence from a protein from <i>Epstein-Barr virus</i>, RNGATFSKGDIEGNF (HCMV US30), the <i>Human herpesvirus-6A</i> protein, the epitope YPAVTTVYPPSSTAK and YDDESWRPLSTVDDH directed against proteins from <i>Human herpesvirus 8</i>, could be found. At 24 months, no matches (using the criteria outlined in materials and methods) between serum CMV epitope recognition and proteomes of other human herpesviruses were found.</p

Differential CMV peptide recognition after HSCT segregates with the CMV status of the donor and recipient.

<p>Comparisons between serum reactivity in the D+ and D− groups and R+ and R− groups reflecting the number of CMV epitopes predicted by the ‘exclusive recognition analysis. Some peptides are uniquely recognized in serum from all individuals in each test group (but never in the control group, D−/R−). The list of epitopes is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089648#pone-0089648-t001" target="_blank">Table 1</a>.</p

Analysis of polyfunctional T cells to previously defined CMV targets and targets defined by peptide array technology.

<p>PBMCs were incubated for 6-2 (IL-2), interferon-γ (IFN-γ) and tumor necrosis factor-alpha, interleukin-17a (IL-17a) productions were measured by intracellular cytokine staining (ICS) on the single-cell level. The cytokine response of one representative individual is shown.</p