Moving from macroscale preparative systems in proteomics to micro- and nanotechnologies offers researchers the ability to deeply profile smaller numbers of cells that are more likely to be encountered in clinical settings. Herein a recently developed microscale proteomic method, microdroplet processing in one pot for trace samples (microPOTS), was employed to identify proteomic changes in ∼200 Barrett's esophageal cells following physiologic and radiation stress exposure. From this small population of cells, microPOTS confidently identified >1500 protein groups, and achieved a high reproducibility with a Pearson's correlation coefficient value of R > 0.9 and over 50% protein overlap from replicates. A Barrett's cell line model treated with either lithocholic acid (LCA) or X-ray had 21 (e.g., ASNS, RALY, FAM120A, UBE2M, IDH1, ESD) and 32 (e.g., GLUL, CALU, SH3BGRL3, S100A9, FKBP3, AGR2) overexpressed proteins, respectively, compared to the untreated set. These results demonstrate the ability of microPOTS to routinely identify and quantify differentially expressed proteins from limited numbers of cells.

Cambridge Oesophagogastric Centre Cambridge University Hospitals NHS Foundation Trust Cambridge CB2 0QQ U K

Department of Biochemistry and Microbiology University of Victoria Victoria BC V8P 5C2 Canada

Environmental Molecular Sciences Laboratory Pacific Northwest National Laboratory Richland Washington 99354 United States

Institute of Genetics and Molecular Medicine University of Edinburgh Edinburgh Scotland EH4 2XR U K

Research Centre for Applied Molecular Oncology Masaryk Memorial Cancer Institute 656 53 Brno Czech Republic

University of Gdansk International Centre for Cancer Vaccine Science ul Kładki 24 80 822 Gdansk Poland

University of Victoria Genome British Columbia Proteomics Centre Victoria BC V8Z 7X8 Canada

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Mann M.; Hendrickson R. C.; Pandey A. Analysis of Proteins and Proteomes by Mass Spectrometry. Annu. Rev. Biochem. 2001, 70, 437–473. 10.1146/annurev.biochem.70.1.437. PubMed DOI

Wasinger V. C.; Cordwell S. J.; Cerpa-Poljak A.; Yan J. X.; Gooley A. A.; Wilkins M. R.; Duncan M. W.; Harris R.; Williams K. L.; Humphery-Smith I. Progress with Gene-Product Mapping of the Mollicutes: Mycoplasma Genitalium. Electrophoresis 1995, 16 (7), 1090–1094. 10.1002/elps.11501601185. PubMed DOI

Zhang Y.; Fonslow B. R.; Shan B.; Baek M.-C.; Yates J. R. Protein Analysis by Shotgun/Bottom-up Proteomics. Chem. Rev. 2013, 113 (4), 2343–2394. 10.1021/cr3003533. PubMed DOI PMC

Mayne J.; Ning Z.; Zhang X.; Starr A. E.; Chen R.; Deeke S.; Chiang C.-K.; Xu B.; Wen M.; Cheng K.; Seebun D.; Star A.; Moore J. I.; Figeys D. Bottom-Up Proteomics (2013–2015): Keeping up in the Era of Systems Biology. Anal. Chem. 2016, 88 (1), 95–121. 10.1021/acs.analchem.5b04230. PubMed DOI

Fagerberg L.; Strömberg S.; El-Obeid A.; Gry M.; Nilsson K.; Uhlen M.; Ponten F.; Asplund A. Large-Scale Protein Profiling in Human Cell Lines Using Antibody-Based Proteomics. J. Proteome Res. 2011, 10 (9), 4066–4075. 10.1021/pr200259v. PubMed DOI

Branca R. M. M.; Orre L. M.; Johansson H. J.; Granholm V.; Huss M.; Pérez-Bercoff Å.; Forshed J.; Käll L.; Lehtiö J. HiRIEF LC-MS Enables Deep Proteome Coverage and Unbiased Proteogenomics. Nat. Methods 2014, 11 (1), 59–62. 10.1038/nmeth.2732. PubMed DOI

Wang G.; Brennan C.; Rook M.; Wolfe J. L.; Leo C.; Chin L.; Pan H.; Liu W.-H.; Price B.; Makrigiorgos G. M. Balanced-PCR Amplification Allows Unbiased Identification of Genomic Copy Changes in Minute Cell and Tissue Samples. Nucleic Acids Res. 2004, 32 (9), e76.10.1093/nar/gnh070. PubMed DOI PMC

Wang N.; Xu M.; Wang P.; Li L. Development of Mass Spectrometry-Based Shotgun Method for Proteome Analysis of 500 to 5000 Cancer Cells. Anal. Chem. 2010, 82 (6), 2262–2271. 10.1021/ac9023022. PubMed DOI

Parapatics K.; Huber M. L.; Lehmann D.; Knoll C.; Superti-Furga G.; Bennett K. L.; Rudashevskaya E. L. Proteomic Analysis of Low Quantities of Cellular Material in the Range Obtainable from Scarce Patient Samples. J. Integr. OMICS 2015, 5 (1), 30–43–43. 10.5584/jiomics.v5i1.172. DOI

Clair G.; Piehowski P. D.; Nicola T.; Kitzmiller J. A.; Huang E. L.; Zink E. M.; Sontag R. L.; Orton D. J.; Moore R. J.; Carson J. P.; Smith R. D.; Whitsett J. A.; Corley R. A.; Ambalavanan N.; Ansong C. Spatially-Resolved Proteomics: Rapid Quantitative Analysis of Laser Capture Microdissected Alveolar Tissue Samples. Sci. Rep. 2016, 6, 6.10.1038/srep39223. PubMed DOI PMC

Coscia F.; Doll S.; Bech J. M.; Mund A.; Lengyel E.; Lindebjerg J.; Madsen G. I.; Moreira J. M. A.; Mann M.. A Streamlined Mass Spectrometry-Based Proteomics Workflow for Large Scale FFPE Tissue Analysis. bioRxiv, Sep 23, 2019, 779009. 10.1101/779009. PubMed DOI

Martin J. G.; Rejtar T.; Martin S. A. Integrated Microscale Analysis System for Targeted Liquid Chromatography Mass Spectrometry Proteomics on Limited Amounts of Enriched Cell Populations. Anal. Chem. 2013, 85 (22), 10680–10685. 10.1021/ac401937c. PubMed DOI

Yamaguchi H.; Miyazaki M.; Honda T.; Briones-Nagata M. P.; Arima K.; Maeda H. Rapid and Efficient Proteolysis for Proteomic Analysis by Protease-Immobilized Microreactor. Electrophoresis 2009, 30 (18), 3257–3264. 10.1002/elps.200900134. PubMed DOI

Myers S. A.; Rhoads A.; Cocco A. R.; Peckner R.; Haber A. L.; Schweitzer L. D.; Krug K.; Mani D. R.; Clauser K. R.; Rozenblatt-Rosen O.; Hacohen N.; Regev A.; Carr S. A. Streamlined Protocol for Deep Proteomic Profiling of FAC-Sorted Cells and Its Application to Freshly Isolated Murine Immune Cells. Mol. Cell. Proteomics 2019, 18 (5), 995–1009. 10.1074/mcp.RA118.001259. PubMed DOI PMC

Gao W.; Li H.; Liu L.; Huang P.; Wang Z.; Chen W.; Ye M.; Yu X.; Tian R. An Integrated Strategy for High-Sensitive and Multi-Level Glycoproteome Analysis from Low Micrograms of Protein Samples. J. Chromatogr. A 2019, 1600, 46–54. 10.1016/j.chroma.2019.04.041. PubMed DOI

Chen W.; Wang S.; Adhikari S.; Deng Z.; Wang L.; Chen L.; Ke M.; Yang P.; Tian R. Simple and Integrated Spintip-Based Technology Applied for Deep Proteome Profiling. Anal. Chem. 2016, 88 (9), 4864–4871. 10.1021/acs.analchem.6b00631. PubMed DOI

Dou M.; Zhu Y.; Liyu A.; Liang Y.; Chen J.; Piehowski P. D.; Xu K.; Zhao R.; Moore R. J.; Atkinson M. A.; Mathews C. E.; Qian W.-J.; Kelly R. T. Nanowell-Mediated Two-Dimensional Liquid Chromatography Enables Deep Proteome Profiling of < 1000 Mammalian Cells. Chem. Sci. 2018, 9 (34), 6944–6951. 10.1039/C8SC02680G. PubMed DOI PMC

Zhu Y.; Scheibinger M.; Ellwanger D. C.; Krey J. F.; Choi D.; Kelly R. T.; Heller S.; Barr-Gillespie P. G. Single-Cell Proteomics Reveals Changes in Expression during Hair-Cell Development. eLife 2019, 8, e50777.10.7554/eLife.50777. PubMed DOI PMC

Shao X.; Zhang X. Design of Five-Layer Gold Nanoparticles Self-Assembled in a Liquid Open Tubular Column for Ultrasensitive Nano-LC-MS/MS Proteomic Analysis of 80 Living Cells. Proteomics 2017, 17 (8), 1600463.10.1002/pmic.201600463. PubMed DOI

Chen Q.; Yan G.; Gao M.; Zhang X. Ultrasensitive Proteome Profiling for 100 Living Cells by Direct Cell Injection, Online Digestion and Nano-LC-MS/MS Analysis. Anal. Chem. 2015, 87 (13), 6674–6680. 10.1021/acs.analchem.5b00808. PubMed DOI

Zhu Y.; Clair G.; Chrisler W. B.; Shen Y.; Zhao R.; Shukla A. K.; Moore R. J.; Misra R. S.; Pryhuber G. S.; Smith R. D.; Ansong C.; Kelly R. T. Proteomic Analysis of Single Mammalian Cells Enabled by Microfluidic Nanodroplet Sample Preparation and Ultrasensitive NanoLC-MS. Angew. Chem., Int. Ed. 2018, 57 (38), 12370–12374. 10.1002/anie.201802843. PubMed DOI PMC

Shao X.; Wang X.; Guan S.; Lin H.; Yan G.; Gao M.; Deng C.; Zhang X. Integrated Proteome Analysis Device for Fast Single-Cell Protein Profiling. Anal. Chem. 2018, 90 (23), 14003–14010. 10.1021/acs.analchem.8b03692. PubMed DOI

Zhu Y.; Piehowski P. D.; Zhao R.; Chen J.; Shen Y.; Moore R. J.; Shukla A. K.; Petyuk V. A.; Campbell-Thompson M.; Mathews C. E.; Smith R. D.; Qian W.-J.; Kelly R. T. Nanodroplet Processing Platform for Deep and Quantitative Proteome Profiling of 10–100 Mammalian Cells. Nat. Commun. 2018, 9 (1), 882.10.1038/s41467-018-03367-w. PubMed DOI PMC

Zhou M.; Uwugiaren N.; Williams S. M.; Moore R. J.; Zhao R.; Goodlett D.; Dapic I.; Paša-Tolić L.; Zhu Y. Sensitive Top-Down Proteomics Analysis of a Low Number of Mammalian Cells Using a Nanodroplet Sample Processing Platform. Anal. Chem. 2020, 92 (10), 7087–7095. 10.1021/acs.analchem.0c00467. PubMed DOI

Xu K.; Liang Y.; Piehowski P. D.; Dou M.; Schwarz K. C.; Zhao R.; Sontag R. L.; Moore R. J.; Zhu Y.; Kelly R. T. Benchtop-Compatible Sample Processing Workflow for Proteome Profiling of < 100 Mammalian Cells. Anal. Bioanal. Chem. 2019, 411 (19), 4587–4596. 10.1007/s00216-018-1493-9. PubMed DOI PMC

Ross-Innes C. S.; Becq J.; Warren A.; Cheetham R. K.; Northen H.; O’Donovan M.; Malhotra S.; di Pietro M.; Ivakhno S.; He M.; Weaver J. M. J.; Lynch A. G.; Kingsbury Z.; Ross M.; Humphray S.; Bentley D.; Fitzgerald R. C. Whole-Genome Sequencing Provides New Insights into the Clonal Architecture of Barrett’s Esophagus and Esophageal Adenocarcinoma. Nat. Genet. 2015, 47 (9), 1038–1046. 10.1038/ng.3357. PubMed DOI PMC

Peters Y.; Al-Kaabi A.; Shaheen N. J.; Chak A.; Blum A.; Souza R. F.; Di Pietro M.; Iyer P. G.; Pech O.; Fitzgerald R. C.; Siersema P. D. Barrett Oesophagus. Nat. Rev. Dis. Primer 2019, 5 (1), 35.10.1038/s41572-019-0086-z. PubMed DOI

Hofmann A. F. The Continuing Importance of Bile Acids in Liver and Intestinal Disease. Arch. Intern. Med. 1999, 159 (22), 2647–2658. 10.1001/archinte.159.22.2647. PubMed DOI

Fitzgerald R. C.; di Pietro M.; O’Donovan M.; Maroni R.; Muldrew B.; Debiram-Beecham I.; Gehrung M.; Offman J.; Tripathi M.; Smith S. G.; Aigret B.; Walter F. M.; Rubin G.; Bagewadi A.; Patrick A.; Shenoy A.; Redmond A.; Muddu A.; Northrop A.; Groves A.; Shiner A.; Heer A.; Takhar A.; Bowles A.; Jarman A.; Wong A.; Lucas A.; Gibbons A.; Dhar A.; Curry A.; Lalonde A.; Swinburn A.; Turner A.; Lydon A.-M.; Gunstone A.; Lee A.; Nambi A.; Ariyarathenam A.; Elden A.; Wilson A.; Donepudi B.; Campbell B.; Uszycka B.; Bowers B.; Coghill B.; de Quadros B.; Cheah C.; Bratten C.; Brown C.; Moorbey C.; Clisby C.; Gordon C.; Schramm C.; Castle C.; Newark C.; Norris C.; A’Court C.; Graham C.; Fletcher C.; Grocott C.; Rees C.; Bakker C.; Paschalides C.; Vickery C.; Schembri D.; Morris D.; Hagan D.; Cronk D.; Goddard D.; Graham D.; Phillips D.; Prabhu D.; Kejariwal D.; Garg D.; Lonsdale D.; Butterworth D.; Clements D.; Bradman D.; Blake D.; Mather E.; O’Farrell E.; Markowetz F.; Adams F.; Pesola F.; Forbes G.; Taylor G.; Collins G.; Irvine G.; Fourie G.; Doyle H.; Barnes H.; Bowyer H.; Whiting H.; Beales I.; Binnian I.; Bremner I.; Jennings I.; Troiceanu I.; Modelell I.; Emmerson I.; Ortiz J.; Lilley J.; Harvey J.; Vicars J.; Takhar J.; Larcombe J.; Bornschein J.; Aldegather J.; Johnson J.; Ducker J.; Skinner J.; Dash J.; Walsh J.; Miralles J.; Ridgway J.; Ince J.; Kennedy J.; Hampson K.; Milne K.; Ellerby K.; Priddis K.; Rainsbury K.; Powell K.; Gunner K.; Ragunath K.; Knox K.; Baseley L.; White L.; Lovat L.; Berney L.; Crockett L.; Murray L.; Westwood L.; Chalkley L.; Leggett L.; Dale L.; Scovell L.; Brooks L.; Saunders L.; Owen L.; Dilwershah M.; Baldry M.; Corcoran M.; Roy M.; Macedo M.; Attah M.; Anson M.-J.; Rutter M.; Wallard M.; Gaw M.; Hunt M.; Lea-Hagerty M.; Penacerrada M.; Bianchi M.; Baker-Moffatt M.; Czajkowski M.; Sleeth M.; Brewer N.; Wooding N.; Todd N.; Millen N.; Zolle O.; Whitehead O.; Ojechi P.; Moore P.; Banim P.; Spellar P.; Bhandari P.; Kant P.; Nixon R.; Russell R.; Roberts R.; Skule R.; West R.; Fox R.; Beesley R.; Gibbins R.; Osborne R.; Thiagarajan S.; Bastiman S.; Warburton S.; Pai S.; Leith-Russell S.; Utting S.; Watson S.; Wytrykowski S.; Singh S.; Malhotra S.; Woods S.; Conway S.; Mateer S.; Macrae S.; Singh S.; Fourie S.; Campbell S.; Parslow-Williams S.; Goel S.; Dellar S.; Jones S.; Knight S.; Mackay-Thomas S.; Mukherjee S.; Allen S.; Henry S.; Evans T.; Leighton T.; Bray T.; Shackleton T.; Santosh V.; Glover V.; Chandraraj V.; Elson W.; Briggs W.; Barron Z.; Khan Z.; Sasieni P. Cytosponge-Trefoil Factor 3 versus Usual Care to Identify Barrett’s Oesophagus in a Primary Care Setting: A Multicentre, Pragmatic, Randomised Controlled Trial. Lancet 2020, 396 (10247), 333–344. 10.1016/S0140-6736(20)31099-0. PubMed DOI PMC

Ross-Innes C. S.; Chettouh H.; Achilleos A.; Galeano-Dalmau N.; Debiram-Beecham I.; MacRae S.; Fessas P.; Walker E.; Varghese S.; Evan T.; Lao-Sirieix P. S.; O’Donovan M.; Malhotra S.; Novelli M.; Disep B.; Kaye P. V.; Lovat L. B.; Haidry R.; Griffin M.; Ragunath K.; Bhandari P.; Haycock A.; Morris D.; Attwood S.; Dhar A.; Rees C.; Rutter M. D.; Ostler R.; Aigret B.; Sasieni P. D.; Fitzgerald R. C. Risk Stratification of Barrett’s Oesophagus Using a Non-Endoscopic Sampling Method Coupled with a Biomarker Panel: A Cohort Study. Lancet Gastroenterol. Hepatol. 2017, 2 (1), 23–31. 10.1016/S2468-1253(16)30118-2. PubMed DOI

Williams S. M.; Liyu A. V.; Tsai C.-F.; Moore R. J.; Orton D. J.; Chrisler W. B.; Gaffrey M. J.; Liu T.; Smith R. D.; Kelly R. T.; Pasa-Tolic L.; Zhu Y. Automated Coupling of Nanodroplet Sample Preparation with Liquid Chromatography–Mass Spectrometry for High-Throughput Single-Cell Proteomics. Anal. Chem. 2020, 92 (15), 10588–10596. 10.1021/acs.analchem.0c01551. PubMed DOI PMC

DEP: Differential Enrichment analysis of Proteomics data version 1.10.0 from Bioconductor, https://rdrr.io/bioc/DEP/ (accessed Jul 4, 2020).

Zhang X.; Smits A. H.; van Tilburg G. B.; Ovaa H.; Huber W.; Vermeulen M. Proteome-Wide Identification of Ubiquitin Interactions Using UbIA-MS. Nat. Protoc. 2018, 13 (3), 530–550. 10.1038/nprot.2017.147. PubMed DOI

test_diff: Differential enrichment test in DEP: Differential Enrichment analysis of Proteomics data, https://rdrr.io/bioc/DEP/man/test_diff.html (accessed Jul 5, 2020).

Ritchie M. E.; Phipson B.; Wu D.; Hu Y.; Law C. W.; Shi W.; Smyth G. K. Limma Powers Differential Expression Analyses for RNA-Sequencing and Microarray Studies. Nucleic Acids Res. 2015, 43 (7), e47–e47. 10.1093/nar/gkv007. PubMed DOI PMC

P’ng C.; Green J.; Chong L. C.; Waggott D.; Prokopec S. D.; Shamsi M.; Nguyen F.; Mak D. Y. F.; Lam F.; Albuquerque M. A.; Wu Y.; Jung E. H.; Starmans M. H. W.; Chan-Seng-Yue M. A.; Yao C. Q.; Liang B.; Lalonde E.; Haider S.; Simone N. A.; Sendorek D.; Chu K. C.; Moon N. C.; Fox N. S.; Grzadkowski M. R.; Harding N. J.; Fung C.; Murdoch A. R.; Houlahan K. E.; Wang J.; Garcia D. R.; de Borja R.; Sun R. X.; Lin X.; Chen G. M.; Lu A.; Shiah Y.-J.; Zia A.; Kearns R.; Boutros P. C. BPG: Seamless, Automated and Interactive Visualization of Scientific Data. BMC Bioinf. 2019, 20 (1), 42.10.1186/s12859-019-2610-2. PubMed DOI PMC

Hulsen T.; de Vlieg J.; Alkema W. BioVenn – a Web Application for the Comparison and Visualization of Biological Lists Using Area-Proportional Venn Diagrams. BMC Genomics 2008, 9 (1), 488.10.1186/1471-2164-9-488. PubMed DOI PMC

Perez-Riverol Y.; Csordas A.; Bai J.; Bernal-Llinares M.; Hewapathirana S.; Kundu D. J.; Inuganti A.; Griss J.; Mayer G.; Eisenacher M.; Pérez E.; Uszkoreit J.; Pfeuffer J.; Sachsenberg T.; Yılmaz Ş.; Tiwary S.; Cox J.; Audain E.; Walzer M.; Jarnuczak A. F.; Ternent T.; Brazma A.; Vizcaíno J. A. The PRIDE Database and Related Tools and Resources in 2019: Improving Support for Quantification Data. Nucleic Acids Res. 2019, 47 (D1), D442–D450. 10.1093/nar/gky1106. PubMed DOI PMC

UniProt , https://www.uniprot.org/ (accessed Jun 25, 2020).

Protein GRAVY, https://www.bioinformatics.org/sms2/protein_gravy.html (accessed Jun 25, 2020).

Budnik B.; Levy E.; Harmange G.; Slavov N. SCoPE-MS: Mass Spectrometry of Single Mammalian Cells Quantifies Proteome Heterogeneity during Cell Differentiation. Genome Biol. 2018, 19 (1), 161.10.1186/s13059-018-1547-5. PubMed DOI PMC

Dou M.; Clair G.; Tsai C.-F.; Xu K.; Chrisler W. B.; Sontag R. L.; Zhao R.; Moore R. J.; Liu T.; Pasa-Tolic L.; Smith R. D.; Shi T.; Adkins J. N.; Qian W.-J.; Kelly R. T.; Ansong C.; Zhu Y. High-Throughput Single Cell Proteomics Enabled by Multiplex Isobaric Labelling in a Nanodroplet Sample Preparation Platform. Anal. Chem. 2019, 91 (20), 13119–13127. 10.1021/acs.analchem.9b03349. PubMed DOI PMC

Weng S. S. H.; Demir F.; Ergin E. K.; Dirnberger S.; Uzozie A.; Tuscher D.; Nierves L.; Tsui J.; Huesgen P. F.; Lange P. F. Sensitive Determination of Proteolytic Proteoforms in Limited Microscale Proteome Samples. Mol. Cell. Proteomics 2019, 18 (11), 2335–2347. 10.1074/mcp.TIR119.001560. PubMed DOI PMC

Kasuga K.; Katoh Y.; Nagase K.; Igarashi K. Microproteomics with Microfluidic-Based Cell Sorting: Application to 1000 and 100 Immune Cells. Proteomics 2017, 17 (13–14), 1600420.10.1002/pmic.201600420. PubMed DOI PMC

Sun J.; Zhang G. L.; Li S.; Ivanov A. R.; Fenyo D.; Lisacek F.; Murthy S. K.; Karger B. L.; Brusic V. Pathway Analysis and Transcriptomics Improve Protein Identification by Shotgun Proteomics from Samples Comprising Small Number of Cells - a Benchmarking Study. BMC Genomics 2014, 15 (Suppl 9), S1.10.1186/1471-2164-15-S9-S1. PubMed DOI PMC

Tanca A.; Abbondio M.; Pisanu S.; Pagnozzi D.; Uzzau S.; Addis M. F. Critical Comparison of Sample Preparation Strategies for Shotgun Proteomic Analysis of Formalin-Fixed, Paraffin-Embedded Samples: Insights from Liver Tissue. Clin. Proteomics 2014, 11 (1), 28.10.1186/1559-0275-11-28. PubMed DOI PMC

Föll M. C.; Fahrner M.; Oria V. O.; Kühs M.; Biniossek M. L.; Werner M.; Bronsert P.; Schilling O. Reproducible Proteomics Sample Preparation for Single FFPE Tissue Slices Using Acid-Labile Surfactant and Direct Trypsinization. Clin. Proteomics 2018, 15 (1), 11.10.1186/s12014-018-9188-y. PubMed DOI PMC

Broeckx V.; Boonen K.; Pringels L.; Sagaert X.; Prenen H.; Landuyt B.; Schoofs L.; Maes E. Comparison of Multiple Protein Extraction Buffers for GeLC-MS/MS Proteomic Analysis of Liver and Colon Formalin-Fixed, Paraffin-Embedded Tissues. Mol. BioSyst. 2016, 12 (2), 553–565. 10.1039/C5MB00670H. PubMed DOI

Tabb D. L.; Vega-Montoto L.; Rudnick P. A.; Variyath A. M.; Ham A.-J. L.; Bunk D. M.; Kilpatrick L. E.; Billheimer D. D.; Blackman R. K.; Cardasis H. L.; Carr S. A.; Clauser K. R.; Jaffe J. D.; Kowalski K. A.; Neubert T. A.; Regnier F. E.; Schilling B.; Tegeler T. J.; Wang M.; Wang P.; Whiteaker J. R.; Zimmerman L. J.; Fisher S. J.; Gibson B. W.; Kinsinger C. R.; Mesri M.; Rodriguez H.; Stein S. E.; Tempst P.; Paulovich A. G.; Liebler D. C.; Spiegelman C. Repeatability and Reproducibility in Proteomic Identifications by Liquid Chromatography–Tandem Mass Spectrometry. J. Proteome Res. 2010, 9 (2), 761–776. 10.1021/pr9006365. PubMed DOI PMC

Delmotte N.; Lasaosa M.; Tholey A.; Heinzle E.; van Dorsselaer A.; Huber C. G. Repeatability of Peptide Identifications in Shotgun Proteome Analysis Employing Off-Line Two-Dimensional Chromatographic Separations and Ion-Trap MS. J. Sep. Sci. 2009, 32 (8), 1156–1164. 10.1002/jssc.200800615. PubMed DOI

Resing K. A.; Meyer-Arendt K.; Mendoza A. M.; Aveline-Wolf L. D.; Jonscher K. R.; Pierce K. G.; Old W. M.; Cheung H. T.; Russell S.; Wattawa J. L.; Goehle G. R.; Knight R. D.; Ahn N. G. Improving Reproducibility and Sensitivity in Identifying Human Proteins by Shotgun Proteomics. Anal. Chem. 2004, 76 (13), 3556–3568. 10.1021/ac035229m. PubMed DOI

Washburn M. P.; Ulaszek R. R.; Yates J. R. Reproducibility of Quantitative Proteomic Analyses of Complex Biological Mixtures by Multidimensional Protein Identification Technology. Anal. Chem. 2003, 75 (19), 5054–5061. 10.1021/ac034120b. PubMed DOI

Magdeldin S.; Yamamoto T. Toward Deciphering Proteomes of Formalin-Fixed Paraffin-Embedded (FFPE) Tissues. Proteomics 2012, 12 (7), 1045–1058. 10.1002/pmic.201100550. PubMed DOI PMC

Maes E.; Broeckx V.; Mertens I.; Sagaert X.; Prenen H.; Landuyt B.; Schoofs L. Analysis of the Formalin-Fixed Paraffin-Embedded Tissue Proteome: Pitfalls, Challenges, and Future Prospectives. Amino Acids 2013, 45 (2), 205–218. 10.1007/s00726-013-1494-0. PubMed DOI

Proungvitaya S.; Klinthong W.; Proungvitaya T.; Limpaiboon T.; Jearanaikoon P.; Roytrakul S.; Wongkham C.; Nimboriboonporn A.; Wongkham S. High Expression of CCDC25 in Cholangiocarcinoma Tissue Samples. Oncol. Lett. 2017, 14 (2), 2566–2572. 10.3892/ol.2017.6446. PubMed DOI PMC

Brychtova V.; Vojtesek B.; Hrstka R. Anterior Gradient 2: A Novel Player in Tumor Cell Biology. Cancer Lett. 2011, 304 (1), 1–7. 10.1016/j.canlet.2010.12.023. PubMed DOI

Suwanmanee G.; Yosudjai J.; Phimsen S.; Wongkham S.; Jirawatnotai S.; Kaewkong W. Upregulation of AGR2vH Facilitates Cholangiocarcinoma Cell Survival under Endoplasmic Reticulum Stress via the Activation of the Unfolded Protein Response Pathway. Int. J. Mol. Med. 2019, 45 (2), 669–677. 10.3892/ijmm.2019.4432. PubMed DOI

Multi-Omic Analysis of Esophageal Adenocarcinoma Uncovers Candidate Therapeutic Targets and Cancer-Selective Posttranscriptional Regulation