200 DETECT – Cost-effective, high-throughput enrichment of low molecular weight proteins in serum proteomics – insights from a CAR-T therapy trial

Background

Global proteomics, despite its efficiency at identifying numerous proteins, is hindered by its bias towards the abundant high molecular weight proteins (HMWPs) due to the more frequent detection of their proteotypic peptides by mass spectrometry.¹ Consequently, the less abundant low molecular weight proteins (LMWPs) are often underrepresented, a challenge that is amplified in serum proteomics.² Existing LMWP detection methods, such as antibody-based techniques and sample lysate pre-fractionation, are both labour-intensive and provide limited protein quantification, rendering them impractical for large-scale proteomics. To address these shortcomings, we developed DETECT (DEep TEChnological Tactics for Serum Proteomics), a streamlined, cost-effective precipitation strategy for LMWP enrichment. DETECT requires minimal specialized equipment, thereby broadening its applicability across most translational research labs.

Methods

Our high-throughput serum proteomics protocol employs 75% acetonitrile precipitation to simultaneously precipitate abundant HMWPs and enrich scarce LMWPs. This method allows for sub-optimal protein precipitation and a single-step delipidation wash, significantly reducing the need for specialized equipment. We validated the optimized protocol against commercially available immunodepleting columns³ using healthy donor serum samples, demonstrating a robust alternative for LMWP enrichment. To evaluate the clinical potential of DETECT, we analysed serum samples from B-cell Acute Lymphoblastic Leukemia or lymphoma patients treated with Kymriah®⁴ at Singapore General Hospital. This analysis sought to reveal differential protein modulation between patient response groups. Complementing the proteome profiling, we conducted a Luminex assay targeting 65 proteins for a comprehensive evaluation of the serum proteome.

Results

DETECT, exhibiting compatibility with mass spectrometry, displayed superior resolution in detecting low-abundant LMWPs compared to commercial methods. When coupled with in-gel protein digestion (excluding the delipidation step), DETECT effectively identifies peptide hormones like Defensin Alpha-1, Galanin Peptides, and Gastric Inhibitory Polypeptide in serum samples. Additionally, we recognized distinct proteomic signatures in the serum of CAR-T therapy responders and non-responders. Notably, an increased presence of chemokines such as CCL8, CCL2, and CXCL12 was observed in non-responders two months post-therapy.

Conclusions

Our innovative DETECT protocol excels in enriching low abundant LMWPs, outperforming commercial methods in serum proteomics. It sheds light on unique proteomic signatures post-CAR-T therapy, thus deepening our understanding of the serum proteome complexity and unearthing distinct immunological responses in non-responders. As a cost-effective, readily adoptable method that requires minimal equipment, DETECT opens possibilities for enhanced serum proteomic studies in most translational research laboratories.

Acknowledgements

The study was funded by a grant from the A*STAR BIOMEDICAL ENGINEERINGPROGRAMME (Project No: C211318003) of the Singapore National MedicalResearch Council (MOH-000323–00, OFYIRG19may-0007) and IAF-PP (HBMS Domain): H19/01/a0/024-SInGapore ImmuNogrAm for Immuno-Oncology (SIGNAL)awarded to J.Y.

References

Aebersold, Ruedi, Matthias Mann. ‘Mass spectrometry-basedproteomics.’ Nature 2003;422(6928):198–207. Issaq, Haleem J, Zhen Xiao, Timothy D Veenstra. ‘Serum andplasma proteomics.’ Chemical reviews 2007;107(8):3601–3620. Bandow, Julia E. ‘Comparison of protein enrichment strategies forproteome analysis of plasma.’ Proteomics 2010;10(7):1416–1425. Philippidis, Alex. ‘Kymriah, First CAR-T Cancer ImmunotherapyApproved by FDA.’ 2017:8–8.

Ethics Approval

This research received ethical approval from the SingHealth CentralisedInstitutional Review Board (iSHaRe Ref. No.: 202203–00014, CIRB Ref No.:2022/2322). Informed consent was obtained from participants, adhering to ICHGCP, the Declaration of Helsinki, and local regulations.