The human secretome – the proteins secreted from human cells

Uhlén, Mathias; Tegel, Hanna; Sivertsson, Åsa; Kuo, Chih‐Chung; Gutierrez, Jahir M.; Lewis, Nathan E.; Dannemeyer, Melanie; Fagerberg, Linn; Malm, Magdalena; Vunk, Helian; Edfors, Fredrik; Hober, Andreas; Kotol, David; Mulder, Jan; Mardinoğlu, Adil; Schwenk, Jochen M.; Nilsson, Peter; Zwahlén, Martin; Takanen, Jenny Ottosson; Feilitzen, Kalle von; Stadler, Charlotte; Lindskog, Cecilia; Pontén, Fredrik; Nielsen, Jens; Palsson, Bernhard Ø.; Volk, Anna-Luisa; Lundqvist, Magnus; Berling, Anna; Svensson, Anne-Sophie; Kanje, Sara; Enstedt, Henric; Afshari, Delaram; Ekblad, Siri; Scheffel, Julia; Katona, Borbala; Vuu, Jimmy; Lindström, Emil; Xu, LanLan; Mihai, Roxana; Bremer, Lucas; Westin, Malin; Muse, Muna; Mayr, Lorenz M.; Knight, Sinéad; Göpel, Sven; Davies, Rick; Varley, Paul; Hatton, Diane; Fields, R. Brett; Voldborg, Bjørn Gunnar; Rockberg, Johan; Schiavone, L. Holmberg; Hober, Sophia

doi:10.1101/465815

Cited by 15 publications

(20 citation statements)

References 27 publications

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“…FE, LF, JR, HT and JMS acknowledge formal links to Atlas Antibodies AB. For capturing, Abs (1) were immobilized onto magnetic color-coded beads (2) and combined into sets of suspension bead arrays (3). A dilution series of EDTA plasma (4a) and protein standard (4b) was performed.…”

Section: Additional Informationmentioning

confidence: 99%

“…Today, the human secretome can be defined by bioinformatics tools annotating our genome based on sequences found in the proteinencoding regions [2]. Using an updated annotation [3], more than 2,600 proteins were defined as the secretome. Of these only about 600 proteins are predicted to be actively secreted to the blood while another 1,000 proteins were localized to the membrane and/or the intracellular space [3].…”

Section: Introductionmentioning

confidence: 99%

“…Using an updated annotation [3], more than 2,600 proteins were defined as the secretome. Of these only about 600 proteins are predicted to be actively secreted to the blood while another 1,000 proteins were localized to the membrane and/or the intracellular space [3].…”

Section: Introductionmentioning

confidence: 99%

“…However, and upon excluding the recent large-scale aptamer studies, only about a third of the currently annotated 2,600 proteins of the secretome [3] can be measured in plasma using other methods. Plasma profiling efforts using shotgun MS, such as those by Mann and co-workers, detected 1,200 proteins [5] in plasma.…”

Section: Introductionmentioning

confidence: 99%

“…Hence it expands on previous workflows that primarily work on selected candidates [7]. Our approach was accelerated by accessing a large resource of antibodies from the Human Protein Atlas (HPA) and full-length proteins generated within the Human Secretome Project [3] (HSP) within the Wallenberg Centre for Protein Research (WCPR). The study was conducted on a multiplexed bead array platform and combined the assessment of antibody pairs using both recombinant proteins and EDTA plasma.…”

Section: Introductionmentioning

confidence: 99%

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Systematic Development of Sandwich Immunoassays for the Plasma Secretome

Haeussler

Bendes

Iglesias

et al. 2019

Preprint

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The plasma proteome offers a clinically useful window into human health and disease. With recent progress made on the development of highly multiplexed immunoassays with high sample throughput, a remaining need is to establish a pipeline for validating the individual proteins that build such bio-signatures by using targeted assays. In order to streamline such efforts, we developed a workflow to build dual binder sandwich immunoassays (SIA) and chose to evaluate this on proteins predicted to be secreted form cells and tissues. Utilizing the multiplexing capacities of the bead array technology, we first screened ~ 1,800 unique antibody pairs against 209 protein targets and collected data from dilution series of recombinant proteins as well as EDTA plasma. Employing 624 unique antibodies from the Human Protein Atlas, we obtained dilution-dependent curves in plasma and concentration-dependent curves of fulllength proteins for 102 (49%) of the targets. For 22 protein assays, the longitudinal, interindividual and technical performance was determined in a set of plasma samples collected from 18 healthy subjects every third month over one year. Lastly, we compared 14 of these assays with SIAs composed of other binders, proximity extension assays and affinity-free targeted mass spectrometry. Our workflow provides a multiplexed approach to screen for SIA pairs that suggests using at least three antibodies per target. This design is applicable for a wider range of targets of the plasma proteome, while the assays can be applied for discovery but also to validate emerging candidates derived from other platforms.(245 words) knowledge or particular interest but rather availability of reagents to conduct this proof-ofconcept study from screening, via validation to the analysis of longitudinal samples.

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Section: Additional Informationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Systematic Development of Sandwich Immunoassays for the Plasma Secretome

Haeussler

Bendes

Iglesias

et al. 2019

Preprint

Self Cite

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Production of trimeric SARS‐CoV‐2 spike protein by CHO cells for serological COVID‐19 testing

Johari

Jaffe

Scarrott

et al. 2020

Biotech & Bioengineering

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We describe scalable and cost‐efficient production of full length, His‐tagged severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike glycoprotein trimer by Chinese hamster ovary (CHO) cells that can be used to detect SARS‐CoV‐2 antibodies in patient sera at high specificity and sensitivity. Transient production of spike in both human embryonic kidney (HEK) and CHO cells mediated by polyethyleneimine was increased significantly (up to 10.9‐fold) by a reduction in culture temperature to 32°C to permit extended duration cultures. Based on these data GS‐CHO pools stably producing spike trimer under the control of a strong synthetic promoter were cultured in hypothermic conditions with combinations of bioactive small molecules to increase yield of purified spike product 4.9‐fold to 53 mg/L. Purification of recombinant spike by Ni‐chelate affinity chromatography initially yielded a variety of co‐eluting protein impurities identified as host cell derived by mass spectrometry, which were separated from spike trimer using a modified imidazole gradient elution. Purified CHO spike trimer antigen was used in enzyme‐linked immunosorbent assay format to detect immunoglobulin G antibodies against SARS‐CoV‐2 in sera from patient cohorts previously tested for viral infection by polymerase chain reaction, including those who had displayed coronavirus disease 2019 (COVID‐19) symptoms. The antibody assay, validated to ISO 15189 Medical Laboratories standards, exhibited a specificity of 100% and sensitivity of 92.3%. Our data show that CHO cells are a suitable host for the production of larger quantities of recombinant SARS‐CoV‐2 trimer which can be used as antigen for mass serological testing.

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Recent Advances in Glycoproteomic Analysis by Mass Spectrometry

2019

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Glycosylation is one of the most common protein modifications and is essential for cells. This modification is exceptionally complex because glycans are highly diverse and can be covalently bound to several amino acid residues in proteins through various configurations. There are two major types of protein glycosylation, i.e., N-linked glycosylation in which glycans are attached to the side chain of asparagine and O-linked glycosylation referring to glycans being bound to the side chains of serine and threonine. 1,2 Glycosylation plays vital roles in cells, including determination of protein folding, trafficking and stability, and regulation of nearly every extracellular activity such as cell-cell communication and cellmatrix interactions. 3,4 Aberrant protein glycosylation is directly related to multiple diseases, including cancer, neurodegenerative disorders, pulmonary diseases, blood disorders, and genetic diseases. 5,6 Due to the importance and complexity of protein glycosylation in biological systems, there is a longstanding interest to develop innovative methods to study glycoproteins and apply them for biomedical research. Investigation of protein glycosylation has become more popular with the development of modern instrumentation and computational methods. According to a PubMed search using the keyword "glycosylation", 16 publications were listed during 1960-1970 while over 20 000 studies were reported in the past 10 years. With the growing interests in protein glycosylation, this trend is expected to continue in the next decades.Mass spectrometry (MS)-based proteomics provides an excellent opportunity to globally analyze proteins and their modifications. [7][8][9][10][11][12][13][14][15][16][17][18][19] Nonetheless, it is still extremely challenging to comprehensively analyze protein glycosylation. 20 Unlike many other modifications with a fixed structure for the modified group, such as phosphorylation, the diversity of glycans makes it more challenging to employ the commonly used database searching methods such as SEQUEST and Mascot to identify glycopeptides in bottom-up proteomics. Lowabundance glycoproteins in complex biological samples are also hindered for detection by many high-abundance nonglycoproteins. Furthermore, glycans can interfere with the fragmentation of the peptide backbone. 20,21 Innovative and effective methods are critical to overcome these hurdles and to allow for comprehensive analysis of glycoproteins using MS.

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The human secretome – the proteins secreted from human cells

Cited by 15 publications

References 27 publications

Systematic Development of Sandwich Immunoassays for the Plasma Secretome

Systematic Development of Sandwich Immunoassays for the Plasma Secretome

Production of trimeric SARS‐CoV‐2 spike protein by CHO cells for serological COVID‐19 testing

Recent Advances in Glycoproteomic Analysis by Mass Spectrometry

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