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Complementarity of different SDS-PAGE gel staining methods for the identification of short open reading frame-encoded peptides

ABSTRACT: Short open reading frame-encoded peptides (SEP) have been identified across all domains of life and are predicted to be involved in many biochemical processes, however, for the vast majority of SEP their biological function is still unknown. Special methodologies have to be used for the mass spectrometric analysis of SEP, because traditional methods of bottom-up proteomics show a bias against small proteins. Here, we investigated the influence of different staining methods for SDS-PAGE gels prior in-gel digestion following LC-MS/MS analysis for the identification of SEP in the archaeon Methanosarcina mazei. In total, we identified 82 SEP with at least one high confidence (FDR<1 %) unique peptide, of which 45 also met a stricter ion series-based quality criteria. The staining methods provided complementary data that allowed for a range of different SEP to be identified. The highest number of SEP were identified in the samples stained with Coomassie brilliant blue. However, the highest quality of the identified SEP was achieved in the samples without staining. These data demonstrate that in-gel digestion is amenable and well suited for the identification of SEP, and that this classical technique may provide further insights into the world of SEP.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Methanosarcina Mazei Go1

SUBMITTER: Andreas Tholey

LAB HEAD: Andreas Tholey

PROVIDER: PXD018269 | Pride | 2023-07-06

REPOSITORIES: Pride

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Project description:Two decades after the introduction of oil-based vaccines in the control of bacterial and viral diseases in farmed salmonids, the mechanisms of induced side effects manifested as intra-abdominal granulomas remain unresolved. Side effects have been associated with generation of auto-antibodies and autoimmunity but underlying profile of the inflammatory and immune response has not been characterized. This study was undertaken with the aim to elucidate the inflammatory and immune mechanisms of granuloma formation at gene expression level associated with high and low side effect (granuloma) indices. Groups of Atlantic salmon parr were injected intraperitoneally with oil-adjuvanted vaccines containing either high or low concentrations of Aeromonas salmonicida or Moritella viscose antigens in order to induce polarized (severe and mild) granulomatous reactions. The established granulomatous reactions were confirmed by gross and histological methods at 3 months post vaccination when responses were known to have matured. The corresponding gene expression patterns in the head kidneys were profiled using salmonid cDNA microarrays followed by validation by real-time quantitative PCR (qPCR). qPCR was also used to examine the expression of additional genes known to be important in the adaptive immune response. Granulomatous lesions were observed in all vaccinated fish. Presence of severe granulomas were associated with a profile of up-regulation of innate immunity-related genes such as complement factors C1q and C6, mannose binding protein, lysozyme C, C-type lectin receptor, CD209, Cathepsin D, CD63, LECT-2, CC chemokine and metallothionein. In addition, IL-17A (Th17) was significantly up-regulated (p=0.009) in the group with severe granulomas as were arginase and IgM. None of the genes directly reflective of Th1 T cell lineage (IFN-γ, CD4) or Th2 (GATA-3) differentiation were differentially expressed. Granulomatous reactions following vaccination with oil-based vaccines in Atlantic salmon has the profile of strong expression of genes related to innate immune responses. The expression of IL-17A suggests an involvement of Th17 T cell lineage and is in conformity with strong infiltration of neutrophils and macrophages into inflamed areas. Arginase upregulation shows that macrophages in these reactions are alternatively activated, indicating a Th2-bias. To what extent the IL-17A profile reflects an autoimmune vaccine-based reaction remains elusive but would be in conformity with previous observations of autoimmune reactions in salmon vaccinated with oil-based vaccines. RNA from 12 head kidney samples (12 fish) per group were pooled into groups of 4. The RNA was amplified prior to hybridization to arrays. Three biological and 2 technical replicates (dye-swapped) were utilized in different combinations to hybridize to 18 arrays using RNA from fish with the least injection-site inflammatory reactions (FO-1) as the reference group. The assignment of microarrays to treatment groups for hybridization was randomised by using a random number generator. To minimize technical variability, target synthesis was done in batches of 6 where all treatment groups were equally represented.

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Complementarity of different SDS-PAGE gel staining methods for the identification of short open reading frame-encoded peptides

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