Incorporating Antibody & Target Information into the HuBMAP Data Portal

Neil Kelleher, Jeannie Camarillo, & HuBMAP Team

@RTI - NU

November 9, 2020

Translating Genes to Proteins �(and their detection)

Align genes with proteins and their antibody information.

Gene 🡪 Protein (‘Target’ or ‘POI’) 🡨 Antibody

Proposal for an “Antibody Registry”: antibody & target information in HuBMAP data portal

• Goal: Include antibody & validation details with each HuBMAP dataset
• Approach: Use Antibody Validation Reports (aVR’s), with informative header
• Version 1.0: Portal UI, & header info on aVR (.pdf format) to be required for data submission
• Provide link to protocols.io method for antibody validation (can be the general methods that were used or custom for each antibody/target)
• Planned for data releases April 2021 and beyond

Elevates organ- and gene-specific operations within HuBMAP:

🡪 data increasingly FAIR: i.e., searchable by gene, protein or antibody

Enhancing ‘Searchability’

Aligns HGNC gene with UniProt Accessions to enable searching and joint visualization

Aquaporin 1

• AQP1
• Found in the kidney proximal tubules
• Allows H₂O to flow in kidney osmosis
• 4 isoforms, some post-translational modifications in UniProt:

He and Yang (2019) Aquaporins in Renal Diseases Int. J. Mol. Sci. 

1. Basic Target Information (aka - header)
2. Validation information

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B.1. Information from Ab vendors.

​

B.2. Laboratory validation

Info. will vary between groups (e.g., immunofluorescence (IF) immunohistochemistry (IHC), gels to show conjugation, etc.

Antibody Validation Report (aVR)

Each group submitting data using antibodies provides Ab info. and validation data.

What aVR header information will be required?

Target Information

UniProt Accession Number:

Target Name:

Antibody Information

RRID:

Antibody Name:

Host Organism:

Clonality:

Vendor:

Catalog Number:

Lot Number:

Recombinant (Y/N):

Organ/Tissue used for validation:

HuBMAP Platform Used:

Protocols.io doi for Validation Protocol:

ORCID ID of submitter:  

THE aVR HEADER

Each group submitting data with antibodies to provide antibody information and validation data.

What aVR header information will be required?

Target Information

UniProt Accession Number: P29972

Target Name: Aquaporin-1

Antibody Information

RRID: AB_626694

Antibody Name: AQP1 Antibody (B-11)

Host Organism: Mouse

Clonality: Monoclonal

Vendor: Santa Cruz

Catalog Number: sc-25287

Lot Number: B2820

Recombinant (Y/N): N

Organ/Tissue used for validation: Human Kidney

HuBMAP Platform Used: IP-MS

Protocols.io doi for Validation Protocol: 10.17504/protocols.io.bpfdmji6

ORCID ID of submitter: 0000-0002-8815-3372

THE HEADER

1. Basic Target Information (aka - header)
2. Validation information

​

B.1. Information from Ab vendors.

​

B.2. Laboratory validation

Info. will vary between groups (e.g., immunofluorescence (IF) immunohistochemistry (IHC), gels to show conjugation, etc.

Antibody Validation Report (aVR)

Vanderbilt aVR on AQP1 (IF)

aVR courtesy of Jeff Spraggins and Elizabeth Neumann at Vanderbilt University

Northwestern RTI – aVR (Mass Spec.)

Two main proteoforms of AQP1 in kidney

Intact mass spectra (MS1) of AQP1 proteoforms:

Unmodified AQP1

PFR5430674

​

Palmitoylated AQP1

PFR5430676

Liquid Chromatography (LC) Run:

mass (Da)

time (min.)

AQP1 proteoform detection

Unmodified AQP1 (17.98 min): MW: 28,377.1618 Da PFR5430674

AQP1 proteoform detection

Palmitoylated AQP1 (21.31 min): MW: 28,615.3835 Da, PFR5430676

Key findings from spatio-molecular proteomics of kidney AQP1

Spatio-

-Molecular (Proteoforms, PFR’s)

• Unmodified proteoform detected in bulk kidney by IP-TDMS
• No glycosylations or phosphorylation (>5% stoichiometry)
• Palmitoylated AQP1 was discovered (~10% abund.; PTM on Cys85?)
• Distribution of these proteoforms in human kidneys, lung, heart & RBCs

AQP1_HUMAN (UniProt accession: P29972)

Protter - visualize proteoforms Omasits et al., Bioinformatics. 2013 Nov 21.

pThr¹⁵⁷ by PKC?*

Palmitoylation, probably on Cys⁸⁵

Zhang, Schwenger, et al. Aquaporin-1 is positively regulated by protein kinase C. J .Biol .Chem. (2007), 282(29), 20933-40

Proposed aVR Implementation Timeline

Future plans for Affinity Reagents Working Group in HuBMAP data portal

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• 2021:
• Develop “quality levels” and icons indicating level of validation to each antibody

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• Better harmonize Ab usage in Consortium

Acknowledgements: RTI Team @NU

Bryon

Drown

Benjamin

Des Soye

Voislav

Blagojevic

Jeannie

Camarillo

Cameron

Lloyd-Jones

Fatma

Butun

Eleonora

Forte

Kelleher Research Group

Proteomics Center of Excellence

Reserve Slides

Human Proteoform PFRs and Knowledgebases

• Proteoform Record Numbers (PFRs) are used to define a proteoform.
• A given sequence, with different modifications, will have different PFRs.
• The Human Proteoform Knowledgebase stores all proteoforms reported in humans.
• Indexed by PFR uniquely assigned to each proteoform in the database.