Phosphopeptide enrichment from complicated peptide mixtures is an essential step for mass spectrometry-based phosphoproteomic studies to reduce sample complexity and ionization suppression effects. Typical methods for enriching phosphopeptides include immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO2) beads, which have selective affinity and can interact with phosphopeptides. In this study, the IMAC enrichment method was compared with the TiO2 enrichment method, using a multi-step enrichment strategy from whole cell lysate, to evaluate their abilities to enrich for different types of phosphopeptides. The peptide-to-beads ratios were optimized for both IMAC and TiO2 beads. Both IMAC and TiO2 enrichments were performed for three rounds to enable the maximum extraction of phosphopeptides from the whole cell lysates. The phosphopeptides that are unique to IMAC enrichment, unique to TiO2 enrichment, and identified with both IMAC and TiO2 enrichment were analyzed for their characteristics. Both IMAC and TiO2 enriched similar amounts of phosphopeptides with comparable enrichment efficiency. However, phosphopeptides that are unique to IMAC enrichment showed a higher percentage of multi-phosphopeptides, as well as a higher percentage of longer, basic, and hydrophilic phosphopeptides. Also, the IMAC and TiO2 procedures clearly enriched phosphopeptides with different motifs. Finally, further enriching with two rounds of TiO2 from the supernatant after IMAC enrichment, or further enriching with two rounds of IMAC from the supernatant TiO2 enrichment does not fully recover the phosphopeptides that are not identified with the corresponding multi-step enrichment.

The contributions of phosphorylation-mediated signaling networks to colon cancer metastasis are poorly defined. To interrogate constitutive signaling alterations in cancer progression, the global phosphoproteomes of the patient-matched SW480 (primary colon tumor origin) and SW620 (lymph node metastasis) cell lines were compared with TiO2 and IMAC phosphopeptide enrichment followed by LC-MS/MS. Network analysis of the significantly altered phosphosites revealed differential regulation in cellular adhesion, mitosis, and mRNA translational machinery. mRNA biogenesis and splicing, transport through the nuclear pores, initiating translation, as well as mRNA stability and degradation were also affected. Though alterations in these processes have been associated with oncogenic transformation, control of mRNA stability has typically not been associated with cancer progression. Notably, the single phosphosite with the greatest relative change in SW620 was Ser2 on eukaryotic initiation factor 2 subunit 2 (eIF2S2), suggesting that SW620 cells translate faster or with greater efficiency than SW480 cells. These broad changes in the regulation of translation also occur without over-expression of eukaryotic initiation factor 4E (eIF4E). The findings suggest that metastatic cells exhibit constitutive changes to the phosphoproteome, and that mRNA stability and translational efficiency may be important targets of deregulation during cancer progression.

Edited by John M. Denu NSD2 is a histone methyltransferase that specifically dimethylates histone H3 lysine 36 (H3K36me2), a modification associated with gene activation. Dramatic overexpression of NSD2 in t(4;14) multiple myeloma (MM) and an activating mutation of NSD2 discovered in acute lymphoblastic leukemia are significantly associated with altered gene activation, transcription, and DNA damage repair. The partner proteins through which NSD2 may influence critical cellular processes remain poorly defined. In this study, we utilized proximity-based labeling (BioID) combined with label-free quantitative MS to identify high confidence NSD2 interacting partners in MM cells. The top 24 proteins identified were involved in maintaining chromatin structure, transcriptional regulation, RNA pre-spliceosome assembly, and DNA damage. Among these, an important DNA damage regulator, poly-(ADP-ribose) polymerase 1 (PARP1), was discovered. PARP1 and NSD2 have been found to be recruited to DNA double strand breaks upon damage and H3K36me2 marks are enriched at damage sites. We demonstrate that PARP1 regulates NSD2 via PARylation upon oxidative stress. In vitro assays suggest the PARylation significantly reduces NSD2 histone methyltransferase activity. Furthermore, PARylation of NSD2 inhibits its ability to bind to nucleosomes and further get recruited at NSD2-regulated genes, suggesting PARP1 regulates NSD2 localization and H3K36me2 balance. This work provides clear evidence of cross-talk between PARylation and histone methylation and offers new directions to characterize NSD2 function in DNA damage response, transcriptional regulation, and other pathways.

(a) Introductory sentence Antibody enrichment and liquid chromatography mass spectrometry (LC-MS) were used to profile lysine acetylation and arginine mono-methylation in triple negative breast cancer patient sera without prior immunodepletion of abundant proteins. (b) Brief description of experiments Triple negative breast cancer (TNBC) is the most aggressive type of breast tumor and there are currently no approved targeted therapies. Better biomarkers are needed for early detection and for therapeutically informative subtyping of this genetically heterogeneous disease. We recently reported a methodology to enrich post-translationally modified peptides from serum (Gu et al, Mol Cell Proteomics 2015) and found acetyl lysine (AcK) and mono-methyl arginine (RMe) to be some of the most abundant post-translational modifications (PTMs) in the sera of cancer patients. This method has the advantage of profiling serum samples without prior depletion of abundant serum proteins, a major limitation of current proteomic methods. To develop a biomarker signature of TNBC, these two PTMs were profiled in the sera of 10 patients with stage I-IIA TNBC and 10 healthy female controls. Serum proteins were trypsin digested prior to immunoaffinity enrichment of the modified peptides with PTM-specific antibodies. The enriched peptides were analyzed by LC-MS and relative abundance of peptides across samples was measured using label-free quantification. (c) Summary of new, unpublished data PTM enrichment quantified hundreds of AcK and RMe sites across samples. AcK levels decreased globally in patients compared to controls, suggestive of increased lysine deacetylase or decreased acetyltransferase activity. Of these, a number of sites were found significantly altered between TNBC patient samples and normal controls at the p<0.05 level by two-sided t-tests, including novel sites not previously reported in the PhosphoSitePlus database or in previous publications. Some of the regulated sites reside on proteins with important immune functions and cancer-related pathways. (d) Statement of conclusions AcK and Rme peptide signatures were identified that correlate with disease status, tumor grade, and stage. Patient follow-up over time will allow for investigation of markers of treatment response and disease progression as well. AcK and Rme are known to affect gene transcription profiles in cancer. Drug molecules targeting multiple families of AcK interacting proteins, including KATs, HDACs, and BET proteins, have entered clinical trials for breast and other cancers. AcK was globally downregulated in the samples surveyed, which suggests that these tumors may be responsive to some of these drug classes. Further analysis will correlate the AcK and RMe profiles to identify a PTM signature of therapeutically vulnerable epigenetic regulators in TNBC. Citation Format: Alissa J. Schunter, Hongbo Gu, Jian Min Ren, Vicky Yang, Matthew P. Stokes. Mass spectrometry-based profiling of lysine acetylation and arginine methylation for biomarker discovery in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2704.