Formalin‐fixed, paraffin‐embedded (FFPE), biobanked tissue samples offer an invaluable resource for clinical and biomarker research. Here we developed a pressure cycling technology (PCT)‐SWATH mass spectrometry workflow to analyze FFPE tissue proteomes and applied it to the stratification of prostate cancer (PCa) and diffuse large B‐cell lymphoma (DLBCL) samples. We show that the proteome patterns of FFPE PCa tissue samples and their analogous fresh frozen (FF) counterparts have a high degree of similarity and we confirmed multiple proteins consistently regulated in PCa tissues in an independent sample cohort. We further demonstrate temporal stability of proteome patterns from FFPE samples that were stored between one to 15 years in a biobank and show a high degree of the proteome pattern similarity between two types of histological regions in small FFPE samples, i.e. punched tissue biopsies and thin tissue sections of micrometer thickness, despite the existence of a certain degree of biological variations. Applying the method to two independent DLBCL cohorts we identified myeloperoxidase (MPO), a peroxidase enzyme, as a novel prognostic marker. In summary, this study presents a robust proteomic method to analyze bulk and biopsy FFPE tissues and reports the first systematic comparison of proteome maps generated from FFPE and FF samples. Our data demonstrate the practicality and superiority of FFPE over FF samples for proteome in biomarker discovery. Promising biomarker candidates for PCa and DLBCL have been discovered.

Serum and plasma contain abundant biological information that reflect the body's physiological and pathological conditions and are therefore a valuable sample type for disease biomarkers. However, comprehensive profiling of the serological proteome is challenging due to the wide range of protein concentrations in serum.

Background : Early screening for colorectal cancer (CRC) is essential to improve its prognosis. Liquid biopsies are increasingly being considered for diagnosing cancer due to low invasiveness and high reproducibility. In addition, circulating extracellular vesicles (crEVs, extracellular vesicles isolated from plasma) expressing tumour‐specific proteins are potential biomarkers for various cancers. Here, we present a data‐independent acquisition (DIA)‐mass spectrometry (MS)‐based diagnostic method for liquid biopsies. Methods : Extracellular vesicles (EVs) were isolated from culture supernatants of human CRC cell lines, and plasma of patients with CRC at different tumour stages, by overnight ultracentrifugation coupled with sucrose density gradient centrifugation. Tumour‐specific EV proteins were prioritized using Tandem Mass Tag (TMT)‐based shotgun proteomics and phosphoproteomics. The results were verified in a second independent cohort and a mouse tumour‐bearing model using Western blotting (WB). The candidate biomarkers were further validated in a third cohort by DIA‐MS. Finally, the DIA‐MS methodology was accelerated to permit high‐throughput detection of EV biomarkers in another independent cohort of patients with CRC and healthy controls. Results : High levels of total and phosphorylated fibronectin 1 (FN1) in crEVs, haptoglobin (HP), S100A9 and fibrinogen α chain (FGA) were significantly associated with cancer progression. FGA was the most dominant biomarker candidate. Analysis of the human CRC cell lines and the mouse model indicated that FGA+ crEVs were likely released by CRC cells. Furthermore, fast DIA‐MS and parallel reaction monitoring (PRM)‐MS both confirmed that FGA+ crEVs could distinguish colon adenoma with an area of curve (AUC) in the receiver operating characteristic (ROC) curve of 0.949 and patients with CRC (AUC of ROC is 1.000) from healthy individuals. The performance outperformed conventional tumour biomarkers. The DIA‐MS quantification of FGA+ crEVs among three groups agreed with that from PRM‐MS. Conclusion : DIA‐MS detection of FGA+ crEVs is a potential rapid and non‐invasive screening tool to identify early stage CRC. Abbreviations: FGA: fibrinogen α chain; CRC: colorectal cancer; crEVs: circulating extracellular vesicles; EV: extracellular vesicles;MS: mass spectrometry; WB: Western blotting; ROC: receiver operating characteristic; PRM: Parallel Reaction Monitoring; GPC1: Glypican‐1; GO: Gene ontology; TEM: transmission electron microscopy; FN1: Fibronectin 1; HP: haptoglobin; TMT: Tandem Mass Tag; LC‐MS/MS: liquid chromatography coupled to tandem mass spectrometry; DIA: data‐independent acquisition; DDA: data‐dependent acquisition; CiRT: Common internal Retention Time standards;AGC: Automatic gain control; AUC: area under curve.

To address the increasing need for detecting and validating protein biomarkers in clinical specimens, mass spectrometry (MS)-based targeted proteomic techniques, including the selected reaction monitoring (SRM), parallel reaction monitoring (PRM), and massively parallel data-independent acquisition (DIA), have been developed. For optimal performance, they require the fragment ion spectra of targeted peptides as prior knowledge. In this report, we describe a MS pipeline and spectral resource to support targeted proteomics studies for human tissue samples. To build the spectral resource, we integrated common open-source MS computational tools to assemble a freely accessible computational workflow based on Docker. We then applied the workflow to generate DPHL, a comprehensive DIA pan-human library, from 1096 data-dependent acquisition (DDA) MS raw files for 16 types of cancer samples. This extensive spectral resource was then applied to a proteomic study of 17 prostate cancer (PCa) patients. Thereafter, PRM validation was applied to a larger study of 57 PCa patients and the differential expression of three proteins in prostate tumor was validated. As a second application, the DPHL spectral resource was applied to a study consisting of plasma samples from 19 diffuse large B cell lymphoma (DLBCL) patients and 18 healthy control subjects. Differentially expressed proteins between DLBCL patients and healthy control subjects were detected by DIA-MS and confirmed by PRM. These data demonstrate that the DPHL supports DIA and PRM MS pipelines for robust protein biomarker discovery. DPHL is freely accessible at https://www.iprox.org/page/project.html?id=IPX0001400000.

The rapid progresses of high-throughput sequencing technology-based omics and mass spectrometry-based proteomics, such as data-independent acquisition and its penetration to clinical studies have generated increasing number of proteomic datasets containing hundreds to thousands of samples. To analyze these quantitative proteomic datasets and other omics (e.g. transcriptomics and metabolomics) datasets more efficiently and conveniently, we present a web server-based software tool ProteomeExpert implemented in Docker, which offers various analysis tools for experimental design, data mining, interpretation and visualization of quantitative proteomic datasets. ProteomeExpert can be deployed on an operating system with Docker installed or with R language environment. The Docker image of ProteomeExpert is freely available from https://hub.docker.com/r/lifeinfo/proteomeexpert. The source code of ProteomeExpert is also openly accessible at http://www.github.com/guomics-lab/ProteomeExpert/. In addition, a demo server is provided at https://proteomic.shinyapps.io/peserver/. Supplementary data are available at Bioinformatics online.