ProteinSeq: High-Performance Proteomic Analyses by Proximity Ligation and Next Generation Sequencing

Abstract

Despite intense interest, methods that provide enhanced sensitivity and specificity in parallel measurements of candidate protein biomarkers in numerous samples have been lacking. We present herein a multiplex proximity ligation assay with readout via realtime PCR or DNA sequencing (ProteinSeq). We demonstrate improved sensitivity over conventional sandwich assays for simultaneous analysis of sets of 35 proteins in 5 µl of blood plasma. Importantly, we observe a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that higher levels of multiplexing are possible. We used ProteinSeq to analyze proteins in plasma samples from cardiovascular disease (CVD) patient cohorts and matched controls. Three proteins, namely P-selectin, Cystatin-B and Kallikrein-6, were identified as putative diagnostic biomarkers for CVD. The latter two have not been previously reported in the literature and their potential roles must be validated in larger patient cohorts. We conclude that ProteinSeq is promising for screening large numbers of proteins and samples while the technology can provide a much-needed platform for validation of diagnostic markers in biobank samples and in clinical use.

Solid-phase proximity ligation assays for individual or parallel protein analyses with readout via real-time PCR or sequencing

“…Using spPLA, levels of the protein GDF--15 have been measured in clinical samples with very good reproducibility, in an investigation lending further support to the role of this protein as a marker for coronary artery disease 12 . A multiplex version of spPLA has been used to simultaneously detect up to 36 proteins in order to identify diagnostic biomarkers for colorectal cancer 13 and cardiovascular disease 12 .…”

“…A multiplex version of spPLA has been used to simultaneously detect up to 36 proteins in order to identify diagnostic biomarkers for colorectal cancer 13 and cardiovascular disease 12 . In addition up to 48 proteins have been analyzed using multiplex spPLA for identification of biomarkers for chronic pain and Alzheimer's disease (Lind et al, in preparation).…”

“…We have demonstrated experimentally that unspecific background increases only minimally when many proteins are detected in parallel, thus allowing assay performance to be maintained with multiplexing, in contrast to the use of sandwich assays 12 . While the homogenous solution phase proximity ligation or extension assays have a simple workflow since there are no washes, spPLA can offer a number of advantages.…”

“…The assay can be used for investigating a single protein species, and it can also be adapted for parallel detection of multiple proteins without an increasing risk of nonspecific reaction products associated with multiplexing. This is possible since the reagents can be designed such that only the correct combinations of PLA probes can give rise to ligated reporter strands 12 . Finally, reporter strand detection via next generation sequencing provides a digital readout for greater assay precision compared to assay readouts where intensities or threshold cycles are used as measures of protein amounts.…”

“…A number of different protocols are available for coupling antibodies to oligonucleotides. Briefly, we have mainly used two approaches: biotin--streptavidin mediated coupling 11 and covalent conjugation by the use of hetero--bi--functional linkers 12 . Streptavidin--linked oligonucleotides as well as antibody--DNA conjugates and commercial conjugation kits can also be purchased from commercial vendors.…”