The emerging landscape of single-molecule protein sequencing technologies

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.This Perspective describes new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell proteomics.</p

From Integrative Omics to Improved Molecular Classification of Head and Neck Cancers

Head and neck squamous cell carcinoma (HNSCC) is a highly heterogeneous disease originating in multiple subsites. Over 60% of HNSCCs are diagnosed at late stages and about 40% recur. Survival and quality of life for these patients is expected to improve upon identification of novel biomarkers, development of new therapies, and obtaining a better understanding of the molecular progression of the disease. Recently, mass spectrometry-based shotgun proteomics technologies have emerged as powerful tools for in-depth proteome characterization, thereby enabling identification of cancer-related proteoforms and surging biomarker discovery efforts. I applied shotgun proteomics towards the identification of novel HNSCC biomarkers and obtaining biologic insight into oropharyngeal carcinoma (OPC) progression. In order to identify secreted biomarker candidates, proteomic analysis of HNSCC cell line secretomes integrated with global gene expression was followed by data mining utilizing published patient tumor expression data and protein annotations. Through subsequent verifications involving IHC and ELISA against patient tissue and sera, respectively, I identified two potential biomarkers (IGFBP7 and PLAU). Their levels were significantly elevated in HNSCC patient sera vs. sera from healthy volunteers. Further evaluation of the clinical utility of these markers is warranted. To gain biologic insight into OPC subtypes, global proteomic analysis of archival patient biopsies with and without HPV was conducted. As a result, 2,633 protein groups were identified, 34 of which were differentially abundant and therefore, may play a role in the distinct clinical behavior of OPC subtypes. Genomic alterations associated with these proteins were determined using The Cancer Genome Atlas; importantly, they were not always predictive of protein abundance. Among the 34 proteins, cortactin is known to mediate radioresistance via the β1-integrin/FAK/cortactin/JNK1/AP-1 pathway. Downstream AP-1 target gene expression analysis revealed HPV-dependent regulation of this pathway, pointing to a putative mechanism for increased radioresistance in HPV-negative vs. HPV-positive OPC. The contribution of this mechanism to therapeutic response requires further evaluation.Ph.D