Nanoparticle-assisted proteomics of whole blood collected through multiple
dried blood spot collection devices
Xiaoyuan Zhou1*, Maedeh Zamani1, Brittany Lee1, Shao-Yung Chen1, Shadi Ferdosi1, Khatereh Motamedchaboki1, Daniel Hornburg1, Alexey Stukalov1, Aaron
Steven Gajadhar1
1Seer, Inc., Redwood City, CA 94065, USA

The Proteograph

Product Suite enables reproducible and deep proteomic analysis​ from whole blood microsampling devices

Introduction

Evaluation of 3 microsampling devices with the Proteograph XT workflow

Microsampling is a methodology for collection and analysis of low-volume biological samples, typically under 50 μL. It
affords several advantages compared to conventional sampling methods: diminished invasiveness, enhanced
convenience, and cost-effectiveness. It allows collecting various biological specimens from diverse sources, finding
widespread application in clinical trials, therapeutic drug monitoring, pharmacogenomics and disease diagnosis.
However, similar to plasma, microsampling of whole blood has a huge dynamic range issue as it has all thecomponents of plasma along with heme and cells. Although in some cases the low volume is beneficial, it also adds
the challenge of sensitivity for a number of assays and quantification.

Whatman®
*4 sample replicates per device

BProteographProtein Group Count

Peptide Group Count

SP100 Automation
Instrument

Centrifugation(1000g 1min)

Peptide preparation

Supernatant(400 μLcollected)

Mass spectrometry analysis

12Particle-matrix assoc.
Protein corona Incubation & Wash

3Protein digestion
Peptide clean-up

Data analysis. Proteomics data was collected in DIA using a 45-minute gradient on Orbitrap Exploris 480 andanalyzed with DIA-NN v1.8.1 in library free mode, with MBR. Peptides were filtered out with q-value<0.01,normalized using global median normalization and rolled up to protein groups using maxRep method.

XT workflow provides unbiased proteomics analyses from whole blood microsampling devices(A) Proteograph with nanoparticles enables approximately 30,000 peptides and 3,000 protein groups quantified ineach of the microsampling devices, deeper than three published references and (B) consistently manifest low
coefficients of variation (CVs) of protein groups below 10% across 4 process replicates.
Direct Digest

Protein corona formation

Proteograph XT
Assay KitCapitainer®

Proteograph XT workflow significantly enhances proteome coverage while
maintaining high reproducibility

A

Proteograph XT workflow

Shake(1200 rpm; 1hrat 4℃)

Sonication(45 sec on/off3× at RT)

Mitra®

In this study, we evaluate the performance of 3 microsampling devices andshow that combining microsampling with nanoparticle-based Seer
Proteograph
XT workflow significantly improves proteome coverage.
Capillary blood were collected from two participants via finger-prick, using
30 μL Mitra devices, 10 μL Capitainer device, and WhatmanTM paper.
Simultaneously, intravenous blood draw was obtained from the same
participant and allocated to Mitra devices.

ProteographProtein
extraction

500 μL water(Optional: PBS, Tris
EDTA, HEPES EDTA)

Proteograph XT workflow is compatible with various protein extraction buffers(A) More than 3000 protein groups were consistently quantified in Mitra + Proteograph XT workflow using different
extraction buffers (water, PBS, TE (10 mM Tris + 1 mM EDTA) and HEPES (20 mM HEPES + 1 mM EDTA) and (B)showed comparable protein groups with an overlap of 3014 (95%) proteins across all five tested buffers.
AB

Lima et al.1
~1800 Proteins(95%)
Whelan et al.2~800 Proteins
Shen et al.3~130 ProteinsProteograph XT workflow enables measurement of low abundant proteins

Capillary blood achieves comparable results to venous blood

ProteographProtein Group CountDirect Digest

B

Participant #1

AC
Log10 HPPP estimated concentration (ng/mL)

A(A) Around 3,200 protein groups were captured in capillary blood using Mitra + Proteograph XT workflow and (B)showed a high overlap (93%) and (C) intensity correlation with the ones from venous blood using Proteograph.

BCParticipant #1
High Concentration

Direct Digest
Proteograph(93%)

Protein log10 (intensity) in venous blood(A) Approximately 3,000 proteins were consistently quantified in Mitra + Proteograph XT workflow across twoparticipants, boosting 3 times more proteins than direct digestion. (B) Mitra + Proteograph captured 92% of theproteins quantified from Proteograph and direct digest. (C) Proteograph yielded more low concentration proteins withmedian concentration at 0.76 ng/mL compared to 8.9 ng/mL of direct digestion based on HPPP database4.

Low Concentration
Protein log10 (intensity) in capillary blood

Proteograph XT workflow enables deep protein coverage (~3,000 proteins) in whole-blood collected throughmicrosampling devices, comparable to venous blood.

Conclusion

Consistent high protein coverage was observed in two participants and multiple different extraction buffers.

The capability for unbiased proteomic exploration at such an unprecedented depth with microsamplingcombined with the Proteograph XT workflow, opens new opportunities for low-volume blood proteomics
including the early discovery of biomarkers and the non-invasive monitoring of health status.

Copyright Seer, Inc., 2024
Seer, Inc., Redwood City, CA 94065, USA | support@seer.bio

References
1 Lima et al., J. Proteome Res. (2022) DOI: 10.1021/acs.jproteome.1c00892
2 Whelan et al., Anal. Chem. (2023) DOI: 10.1021/acs.analchem.3c01189
3 Shen et al., Nat. Biomed. Eng. (2023) DOI: 10.1038/s41551-022-00999-8
4 Schwenk et al., J. Proteome Res. (2017) DOI: 10.1021/acs.jproteome.7b00467