Current Methods of Post-Translational Modification Analysis and Their Applications in Blood Cancers

Abstract

Post-translational modifications (PTMs) add a layer of complexity to the proteome through the addition of biochemical moieties to specific residues of proteins, altering their structure, function and/or localization. Mass spectrometry (MS)-based techniques are at the forefront of PTM analysis due to their ability to detect large numbers of modified proteins with a high level of sensitivity and specificity. The low stoichiometry of modified peptides means fractionation and enrichment techniques are often performed prior to MS to improve detection yields. Immuno-based techniques remain popular, with improvements in the quality of commercially available modification-specific antibodies facilitating the detection of modified proteins with high affinity. PTM-focused studies on blood cancers have provided information on altered cellular processes, including cell signaling, apoptosis and transcriptional regulation, that contribute to the malignant phenotype. Furthermore, the mechanism of action of many blood cancer therapies, such as kinase inhibitors, involves inhibiting or modulating protein modifications. Continued optimization of protocols and techniques for PTM analysis in blood cancer will undoubtedly lead to novel insights into mechanisms of malignant transformation, proliferation, and survival, in addition to the identification of novel biomarkers and therapeutic targets. This review discusses techniques used for PTM analysis and their applications in blood cancer research.

Keywords: acetylation; blood cancer; leukemia; lymphoma; multiple myeloma; myeloproliferative neoplasms; phosphorylation; post-translational modifications; sumoylation; ubiquitination.

Figure 1

Post-translational modifications (PTMs) within the mammalian cell. This figure illustrates some of the most well-known PTMs and their functions within the cell. PTMs are found throughout the cell from the plasma membrane to the nucleosomes present within the nucleus. PTMs play crucial roles in almost all cellular processes including the cell cycle, degradation, apoptosis, cell signaling, transcription, etc. Different proteins modified by the same PTM will not always yield the same response, demonstrating the diverse functions of PTMs within the cell. JAK, Janus kinase; STAT, signal transducer and activator of transcription; P, phosphate group; G, glycosyl group; Ub, ubiquitin; SUMO, small ubiquitin-like modifier; Ac, acetyl group; Me, methyl group; Val, valine; Met, methionine; Cys, cysteine; Tyr, tyrosine; Glu, glutamic acid; Lys, lysine.

Figure 2

Schematic workflow illustrating analytical techniques used in the analysis of post-translational modifications. Proteomic techniques used for PTM analysis can be divided into antibody-based analysis or mass spectrometry (MS)-based analysis. Protein microarrays, immunohistochemistry and Western blot analysis demonstrate widespread popularity as efficient tools for antibody-based PTM analysis. MS-based analysis is a powerful technique in the study of PTMs. This figure outlines the steps involved in the preparation of crude samples for MS and the transformation of MS data into interpretable results.

Figure 3

Bottom-up proteomics workflow for PTM analysis and subsequent validation. Bottom-up proteomic analyses are often applied for PTM-focused studies. This workflow can be adapted based on the PTM of interest through the selection of PTM-specific enrichment techniques. The examples provided in the figure (red font) highlight potential adaptations of this approach for blood cancer research. Middle-down and top-down proteomic approaches for PTM analysis are gaining popularity and may also present as useful techniques for future studies in blood cancer research [180,181]. FASP, filter-aided sample preparation; TMT, tandem mass tag; SCX, strong cation exchange chromatography; IMAC, immobilized metal ion affinity chromatography; MOAC, metal oxide affinity chromatography; TUBEs, tandem ubiquitin-binding entities, UBDs, ubiquitin-binding domains; SUBEs, SUMO-binding entities; HILIC, hydrophilic interaction liquid chromatography; COFRADIC, combined fractional diagonal chromatography. Created using BioRender.com.

Figure 4

Targeting of protein modifications by clinically used blood cancer therapeutics. Ruxolitinib, midostaurin, gilteritinib, imatinib, dasatinib, nilotinib, bosutinib, ponatinib, ibrutinib and idelalisib are kinase inhibitors that inhibit the phosphorylation and activation of key proteins of oncogenic signaling pathways, promoting growth arrest and apoptosis in cancer cells. ATRA therapy for the treatment of APL induces SUMOylation-dependent polyubiquitination and proteasomal degradation of the fusion oncoprotein PML-RARA. The immunomodulatory drugs, lenalidomide, thalidomide and pomalidomide bind Cereblon, the substrate adaptor of the CRL4^CRBN E3 ubiquitin ligase, modulating its substrate specificity. Bortezomib, carfilzomib and ixazomib target the 26S proteasome, blocking the degradation of polyubiquitinated regulatory proteins and inducing the unfolded protein response resulting in apoptosis. Panobinostat, vorinostat, belinostat and romidepsin are HDAC inhibitors that block the deacetylation activity of HDACs and exert their anti-tumor activity through modulating the acetylation status of a variety of histone and non-histone proteins. JAK2^V617F, Janus kinase 2 V617F; STAT, signal transducer and activator of transcription; SHC, Src homology and Collagen; GRB2, growth factor receptor-bound protein 2; SOS, Son of sevenless; RAF, RAF proto-oncogene serine/threonine-protein kinase; MEK, Mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase; FLT3-ITD, fms like tyrosine kinase 3 – internal tandem duplications; PI3K, phosphatidylinositol 3-kinase; MDM2, mouse double minute 2 homolog; IKK, IκB kinase; Bad, BCL2 associated agonist of cell death; NF-κB, Nuclear factor kappa B; Bcl-xL, B-cell lymphoma-extra large; GAB2, GRB2 associated binding protein 2; BCR-ABL, breakpoint cluster region-proto-oncogene tyrosine-protein kinase; CBL, E3 ubiquitin-protein ligase CBL; CRK, Proto-oncogene c-Crk; CRKL, Crk-like protein; CAS, Crk-associated substrate; FAK, focal adhesion kinase; BCR, B-cell receptor; Lyn, tyrosine-protein kinase Lyn; SYK, tyrosine-protein kinase SYK; BTK, Bruton’s tyrosine kinase; BLNK, B-cell linker protein; PLC, phospholipase C; DAG, diacyl glycerol; IP₃, inositol 1,4,5-trisphosphate; NFAT, nuclear factor of activated T-cells; PML-RARA, promyelocytic leukemia/retinoic acid receptor alpha; ATRA, all-trans retinoic acid; Ubc9, SUMO-conjugating enzyme UBC9; RNF4, ring finger protein 4; CUL4A, cullin 4A; DDB1, DNA damage binding protein 1; CRBN, cereblon; ROC1, regulator of cullins-1; Len, lenalidomide; JNK, c-Jun N-terminal kinase (JNK); IκB, inhibitor of nuclear factor kappa B; HIF-1α, hypoxia-inducible factor 1-alpha; HSP90, heat shock protein 90; P, phosphorylation; S, sumoylation; Ub, ubiquitination; Ac, acetylation.