Quantitative Proteomics and Metabolomics Reveal Biomarkers of Disease as Potential Immunotherapy Targets and Indicators of Therapeutic Efficacy

Abstract

Quantitative mass spectrometry (MS) continues to deepen our understanding of the immune system, quickly becoming the gold standard for obtaining high-throughput, quantitative data on biomolecules. The development of targeted and multiplexed assays for biomarker quantification makes MS an attractive tool both for diagnosing diseases and for quantifying the effects of immunotherapeutics. Because of its accuracy, the use of MS for identifying biomarkers of disease reduces the potential for misdiagnosis and overtreatment. Advances in workflows for sample processing have drastically reduced processing time and complexities due to sample preparation, making MS a more accessible technology. In this review, we present how recent developments in proteomics and metabolomics make MS an essential component of enhancing and monitoring the efficacy of immunotherapeutic treatments.

Keywords: Quantitative mass spectrometry; immunotherapy targets; metabolomics; proteomics.

Figure 1

Type of cancer immunotherapy. The arsenal of cancer immunotherapy has grown to feature four main types. Effector cellular transfer can utilize lymphocytes gathered from tumor tissue (TILs) or cultured with tumor antigens (antigen specific T cells), or genetically modified lymphocytes. Oncolytic viruses are viruses that preferentially target and kill cancer cells, and are more recently being engineered to introduce immuno-stimulating transgenes. Cancer vaccines prime the immune system to eliminate cancer from the recognition of cancer antigens. Antibody therapies include direct targeting of tumor antigens, as well as by stimulating an antitumor response by blocking inhibitory signals and/or providing additional stimulation.

Figure 2

Schematic of sample flow through MS. A typical and general mass spectrometer set-up includes the introduction of a sample through an electrospray ionization source to produce protonated molecules. These molecules are then directed through an electromagnetic field to facilitate separation by mass-to-charge (m/z) ratio. A detector records information about the molecules passing through the mass analyzer that can be evaluated further through comparison to molecular mass libraries.

Figure 3

Peptide Labeling Methods in Proteomics. Sample preparation for injection onto the mass spectrometer can be done in multiple different manners to provide optimal quantitation. A) Chemical labeling can be done using iTRAQ- or TMT- isotope labels that bind to samples individually and are identified by small mass shifts during MS2. This type of labeling allows relative quantification and multiplexing of samples to enhance the high-throughput capacities of the mass spectrometer. B) Metabolic labeling involves the use of isotope-labeled amino acids in cell media to promote incorporation of isotopically-labeled amino acids in proteins formed by the cells during growth. This method also enables multiplexing of samples and subsequent identification of each using MS1 analysis. C) Label-free quantitation involves MS analysis without the addition of any label but cannot be used when interested in relative quantification or multiplexing.

Figure 4

Schematic overview of the use of hybrid tracers in theranostic applications A) Components that make up the hybrid tracer used to target CXCR4: a Cy5-fluorescent dye, a DTPA-chelate and the CXCR4 targeting peptide Ac-TZ14011. After functionalization with either a radioisotope (radiolabel; yellow) or a non-ionizing lanthanide isotope (blue), this tracer also becomes of value for respectively nuclear medicine (NM) or mass spectrometry (MS) based applications. B) In vitro this tracer can be used in fluorescence (FL)- (red) and MS-based cytometry and imaging studies. C) In vivo NM-based imaging studies can be complemented with NM- or MS-based analysis of uptake levels in tissues and D) ex vivo FL- and MS-based imaging could be used to evaluate the degree and heterogeneity of tissue staining following in vivo tracer administration .

Figure 5

Checkpoint inhibitors targeting the PD-1 and PD-L1 interaction between T cells and tumor cells. An interaction between PD-L1 expressed on tumor cells and PD-1 expressed on T cells often acts as a suppressor for T-cells activated against signs of disease. This schematic illustrates the use of anti-PD-1 and anti-PD-L1 antibodies, known as checkpoint inhibitors, to reduce the potential of this suppression and enable the T-cells to act against cancerous cells.

Figure 6

The -omics cascade. The -omics cascade follows the journey from genotype to phenotype. While genomics, transcriptomics, and proteomics are essential to understanding disease, metabolomics is more closely linked to phenotype and metabolites are not readily subject to ex post facto modifications.