Protein Quantification in Clinical Samples

Changes in the biological states of cell populations can cause disease phenotypes. Every cell produces proteins that mediate those phenotypes. “In the central dogma of genetics, proteins represent real-time biology as they are dynamic and actionable targets in both health and disease,” explains Katarina Hornaeus, Global Product Manager at Olink Proteomics. “Moreover, proteins represent the impact of environmental and lifestyle factors to cellular phenotypes as cells respond to external stimuli.” Alterations to protein abundance and behavior can thus help clinicians diagnose disease and ensure timely treatment.

As proteins play important roles in a cell’s biological state, they become biomarkers ready to be discovered as targets for drug development.¹ Identifying the biomarkers for different disease states, when combined with a deeper understanding of disease mechanisms, can “help provide patients the correct treatment in early disease states and improve clinical outcomes,” adds Hornaeus. Measuring protein abundances thus represents an important aspect in the study and diagnosis of human disease. In response, many methods to identify and quantify proteins have been developed, each of which serves to identify and validate novel disease biomarkers.

Mass spectrometry (MS) for protein quantification

MS is a foundational tool for characterizing a protein’s primary structure. It relies on measuring the mass-to-charge (m/z) ratios of ionized molecules to elucidate a protein’s peptide sequence.² Modifications to existing MS pipelines have enhanced its ability to detect changes in protein structure. For one, electrospray ionization (ESI-MS)³ and top-down mass spectrometry (TD-MS)⁴ have allowed native proteins to be retained as they are ionized in the mass spectrometer. This enables the characterization of not only protein abundances but also the abundance of protein species with specific post-translation modifications that add another layer to a cell’s phenotype.⁵

Other MS modifications enable accurate protein quantification. In stable isotope labeling by amino acids in cell culture (SILAC), two cell populations are labeled with either a light isotope or a heavy isotope to produce light and heavy amino acids.⁶ This approach enables absolute quantification with internal standards, but these assays can only be implemented with in vitro cell cultures.⁷ Isobaric tags for relative and absolute quantification (iTRAQ) enable labeling the N-terminus and side chains of primary amines with a single isotope species in peptides and proteins.⁸ The presence of reporter ions generated after fragmentation is then used to quantify protein abundances.⁹

Despite advances to protocols in MS, Hornaeus notes its limitations in protein quantification. “While mass spectrometry works excellently for protein discovery in cell and tissue lysates, extensive sample preparations are needed to detect low-abundance proteins in complex clinical specimens such as plasma, serum, or cerebrospinal fluid (CSF). This can introduce variance and bias into studies, complicating protein quantification efforts. Additionally, mass spectrometers require substantial space and maintenance, reducing the scalability for protein quantification,” she notes.

Antibody-based protein quantification—multiplex immunoassays

Immunoassays use antibodies that quantify target molecules by monitoring the binding of antibodies specific to these targets. The first immunoassays measured the activity of a reporter enzyme bound to a detection antibody through enzyme-linked immunosorbent assays (ELISA).¹⁰ However, ELISAs can only assay one analyte per reaction, reducing the scalability for assessing proteins as disease biomarkers. Multiplex immunoassays address this using distinct, color-coded beads that capture distinct analytes in solution and use a fluorescent dye for detection. This allows multiple proteins to be quantified concurrently within a single reaction well to increase scalability and reduce reagent costs. Such assays have been used to quantify growth factors, trophic factors, and other proteins that play a role in stem cell communication, survival, proliferation, differentiation, and death.¹¹

Irrespective of the kind of immunoassay, cross-reactivity with the antibodies can take place. Antibodies can bind to molecules other than the target protein, adversely affecting accuracy and reproducibility. “The plex-grade or composition of the panel also impacts quantification through multiplex immunoassays. Furthermore, wash steps after the introduction of antibodies can increase the risk of losing relevant information,” Hornaeus notes. To address these shortcomings, Olink Proteomics has developed the proximity extension assay (PEA). Instead of employing a single antibody, PEA uses two matched antibodies, each linked with a DNA-encoded oligonucleotide tag that can hybridize with each other. Once hybridized, the DNA barcode sequences can be amplified and quantified with quantitative PCR (qPCR) or next-generation sequencing (NGS).

The PEA pipeline enhances specificity and sensitivity, introducing the ability to quantify low-abundance proteins at a high-throughput rate. Hornaeus attributes the increased sensitivity of PEAs to “a workflow free of sample pre-processing, PCR pre-amplification, and the use of qPCR and NGS-based techniques to quantify protein abundances.” In support of this, PEA complemented MS-based approaches by measuring 728 plasma proteins with a broad dynamic range and sensitivity down to pg/mL levels.¹² This approach was also used to identify 11 protein signatures that distinguished patients with cervical cancer at the time of diagnosis.¹³

PEA can also quantify nearly 3000 proteins in 96 samples within a single run with the help of automated sample preparation. Hornaeus attributes the flexibility of their Flex product as follows, “Our Flex product contains a library of approximately 200 pre-validated protein biomarker assays from which the customer can freely mix and match 15–21 of these to build their own Flex panel. Each of the validations includes controlling for sensitivity, specificity, precision, linearity, and biological range.”

Conclusion

Biomedical research has morphed into a desire to delve deeper into the molecular mechanisms that underlie disease. Proteins are among the key mediators of these mechanisms, driving cellular phenotypes and dictating health and disease states. Many approaches for protein quantification have been developed to study the role of proteins in disease further. MS-based methods help identify novel proteins but struggle in achieving high-throughput and absolute quantification. Multiplex immunoassays address issues with scalability, but cross-reactivity can reduce the accuracy of protein quantification. The PEA technologies developed by Olink Proteomics address both issues, enabling protein quantification at a broad range of detection and achieving high sensitivity and specificity. With technologies like Olink Flex emerging, quantitative proteomics is taking massive steps forward toward identifying and validating novel protein biomarkers of disease and health.

References

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