Dataset Information

B-cell-specific MYD88 L252P expression causes a premalignant gammopathy resembling IgM MGUS

ABSTRACT: A highly recurrent, somatic L265P mutation in the TIR domain of the signaling adapter MYD88 constitutively activates NF-kB. It occurs in nearly all human patients with Waldenström’s macroglobulinemia (WM), a B cell malignancy caused by IgM-expressing cells. Here we introduced an inducible leucine to proline point mutation into the mouse Myd88 locus, at the orthologous position L252P. When the mutation was introduced early during B cell development, B cells developed normally. However, IgM-expressing plasma cells accumulated with age in spleen and bone, leading to more than 20-fold elevated serum IgM titers. When introduced into germinal center B cells in the context of an immunization, the Myd88L252P mutation caused prolonged persistence of antigen-specific serum IgM and elevated numbers of antigen-specific IgM plasma cells. Myd88L252P-expressing B cells switched normally, but plasma cells expressing other immunoglobulin isotypes did not increase in numbers, implying that IgM expression may be required for the observed cellular expansion. In order to test whether the Myd88L252P mutation can cause clonal expansions, we introduced it into a small fraction of CD19-positive B cells. In this scenario, five out of five mice developed monoclonal IgM serum paraproteins accompanied by an expansion of clonally related plasma cells that expressed mostly hypermutated VDJ regions. Taken together, our data suggest that the Myd88L252P mutation is sufficient to promote aberrant survival and expansion of IgM-expressing plasma cells which in turn can cause IgM monoclonal gammopathy of undetermined significance (MGUS), the premalignant condition that precedes WM. References: 1. Shevchenko A, Tomas H, Havli J, Olsen JV, Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc (2006) 1:2856–2860. doi:10.1038/nprot.2006.468, 2. Rappsilber J, Mann M, Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc (2007) 2:1896–1906. doi:10.1038/nprot.2007.261, 3. Cox J, Neuhauser N, Michalski A, Scheltema RA, Olsen JV, Mann M. Andromeda: A Peptide Search Engine Integrated into the MaxQuant Environment. J Proteome Res (2011) 10:1794–1805. doi:10.1021/pr101065j, 4. Cox J, Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol (2008) 26:1367–1372. doi:10.1038/nbt.1511.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Plasma Cell

DISEASE(S): Waldenstroem's Macroglobulinemia

SUBMITTER: Oliver Popp

LAB HEAD: Klaus Rajewsky

PROVIDER: PXD017292 | Pride | 2020-11-06

REPOSITORIES: Pride

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			Action	DRS
	Gonzo_20190820_OP_MM_KrSch_Mspikes_Sample02_51_IgM.raw	Raw
	Gonzo_20190820_OP_MM_KrSch_Mspikes_Sample03_47_IgM.raw	Raw
	Gonzo_20190820_OP_MM_KrSch_Mspikes_Sample07_48_IgM.raw	Raw
	Gonzo_20190820_OP_MM_KrSch_Mspikes_Sample11_49_IgM.raw	Raw
	Gonzo_20190820_OP_MM_KrSch_Mspikes_Sample13_50_IgM.raw	Raw

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Project description:We quantified the protein abundances of Mycoplasma pneumoniae (Mpn), a genome-reduced organism, by combining selected reaction monitoring of a reduced set of labeled peptides and label-free shotgun mass spectrometry. A set of 77 labeled peptides were synthesized and used as internal standard peptides to precisely and quantitatively measure the absolute concentrations of proteins in a tandem mass spectrometer [1]. The heavy peptides were chosen based on tryptic peptides of endogenous proteins previously observed in mass spectrometry experiments and that covered more than a 1000-fold range of protein concentrations. Three time points of Mpn growth curve (48, 72 and 96 hours) and the heavy peptides were injected per triplicates in order to calculate the endogenous amount of each peptide (single reference point quantitation) [2], resulting in the absolute quantification of 73 reference proteins. A calibration curve between the absolute amount of reference proteins and the proteome-wide protein intensities allowed the estimation of absolute protein abundances for all detected proteins [3]. In order to further improve the quantification, four additional label-free mass spectrometry datasets of Mpn [4] using different protein extraction methods, from a previous project, were combined with the original dataset, which allowed to quantify the abundances of 528 proteins (75% of the proteome of Mpn). 1. Gerber SA, Rush J, Stemman O, Kirschner MW, Gygi SP. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc Natl Acad Sci U S A. 2003;100: 6940–6945. doi:10.1073/pnas.0832254100 2. Campbell J, Rezai T, Prakash A, Krastins B, Dayon L, Ward M, et al. Evaluation of absolute peptide quantitation strategies using selected reaction monitoring. Proteomics. 2011;11: 1148–1152. doi:10.1002/pmic.201000511 3. Schmidt A, Kochanowski K, Vedelaar S, Ahrné E, Volkmer B, Callipo L, et al. The quantitative and condition-dependent Escherichia coli proteome. Nat Biotechnol. 2016;34: 104–110. doi:10.1038/nbt.3418 4. Miravet-Verde S, Ferrar T, Espadas-García G, Mazzolini R, Gharrab A, Sabido E, et al. Unraveling the hidden universe of small proteins in bacterial genomes. Mol Syst Biol. 2019;15: e8290. doi:10.15252/msb.20188290 5. Yus E, Maier T, Michalodimitrakis K, van Noort V, Yamada T, Chen W-H, et al. Impact of genome reduction on bacterial metabolism and its regulation. Science. 2009;326: 1263–1268. doi:10.1126/science.1177263 6. Wiśniewski JR, Zougman A, Nagaraj N, Mann M. Universal sample preparation method for proteome analysis. Nat Methods. 2009;6: 359–362. doi:10.1038/nmeth.1322 7. Picotti P, Bodenmiller B, Mueller LN, Domon B, Aebersold R. Full dynamic range proteome analysis of S. cerevisiae by targeted proteomics. Cell. 2009;138: 795–806. doi:10.1016/j.cell.2009.05.051 8. Martens L, Van Damme P, Van Damme J, Staes A, Timmerman E, Ghesquière B, et al. The human platelet proteome mapped by peptide-centric proteomics: a functional protein profile. Proteomics. 2005;5: 3193–3204. doi:10.1002/pmic.200401142 9. Desiere F, Deutsch EW, King NL, Nesvizhskii AI, Mallick P, Eng J, et al. The peptideatlas project. Nucleic Acids Res. 2006;34: D655–D658. Available: https://academic.oup.com/nar/article-abstract/34/suppl_1/D655/1132687 10. Lange V, Picotti P, Domon B, Aebersold R. Selected reaction monitoring for quantitative proteomics: a tutorial. Mol Syst Biol. 2008;4: 222. doi:10.1038/msb.2008.61 11. MacLean B, Tomazela DM, Shulman N, Chambers M, Finney GL, Frewen B, et al. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics. 2010;26: 966–968. doi:10.1093/bioinformatics/btq054 12. Perkins DN, Pappin DJ, Creasy DM, Cottrell JS. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis. 1999;20: 3551–3567. doi:10.1002/(SICI)1522-2683(19991201)20:18<3551::AID-ELPS3551>3.0.CO;2-2 13. Elias JE, Gygi SP. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat Methods. 2007;4: 207–214. doi:10.1038/nmeth1019

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B-cell-specific MYD88 L252P expression causes a premalignant gammopathy resembling IgM MGUS

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