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Qualitative Analysis of short open reading frame encoded proteins from Methanosarcina mazei under stress conditions via bottom up and top down proteomics

ABSTRACT: In the present work we have analysed Methanosarcina mazei grown under different stressor conditions via combined top-down and bottom-up proteomic workflows to determine which SEP are differenitally present in responce to stress conditions

INSTRUMENT(S):

ORGANISM(S): Methanosarcina Mazei Go1

SUBMITTER: Andreas Tholey

LAB HEAD: Andreas Tholey

PROVIDER: PXD055745 | Pride | 2024-09-24

REPOSITORIES: Pride

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			Action	DRS
	20211112_EH_01_N_3ul_3hr.raw	Raw
	20211112_EH_02_N_3ul_3hr.raw	Raw
	20211112_EH_03_Nstat_3ul_3hr.raw	Raw
	20211112_EH_04_Nstat_3ul_3hr.raw	Raw
	20211112_EH_05_42C_3ul_3hr.raw	Raw

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Quantitative analsysis of short open reading frame encoded proteins from Methanosarcina mazei under stress conditions via bottom up proteomics

Project description:In the present work we have analysed Methanosarcina mazei grown under different stressor conditions via quantitative bottom-up proteomic workflows to determine which SEP are differenitally abundant in responce to stress conditions

2024-09-24 | PXD055748 | Pride

Exploring the hidden world of Methanosarcina mazei: Unravelling the secrets of its small proteome and its nitrogen-dependent regulation

Project description:The mesophilic methanogenic archaeal model organism Methanosarcina mazei strain Gö1, is crucial for climate and environmental research due to its ability to produce methane from H2 plus CO2, acetate, and methylamines. Here, we established the first Ribo-seq protocol for M. mazei strain Gö1 under two growth conditions (nitrogen sufficiency (+N) versus nitrogen limitation (-N)). Translation of 93 previously annotated and 311 novel small open reading frames (sORFs), coding for proteins ≤ 70 amino acids in length was predicted with high confidence based on Ribo-seq data. Epitope tagging followed by immunoblotting analysis confirmed the translation of 12 out of 15 selected novel sORFs, the remaining three were validated by LC-MS-MS analysis. In total, LC-MS-MS analysis validated translation for 60 annotated sORFs and 26 novel sORFs. A comprehensive differential expression analysis revealed that 29 of 311 novel sORFs were differentially regulated in response to nitrogen availability at the transcriptional and 49 at the translational level. Several reported sRNAs are now emerging as dual-functional sRNAs, including sRNA154, the central regulatory sRNA in regulation of nitrogen metabolism. Numerous of the novel sORFs identified in this study are conserved in Methanosarcina species, some of which showed a phenotype when overproduced, pinpointing important physiological functions. Overall, the comprehensive analysis opens a new avenue to start elucidating the function(s) of small proteins in M. mazei.

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Multidimensional separation schemes enhance the identification and molecular characterization of low molecular weight proteomes and short open reading frame encoded peptides in top-down proteomics

Project description:In the present work we developed a sample preparation approach for the combined bottom-up and top-down proteomics analysis of small open reading frame encoded proteins (SEP). Key improvements were made by the application of solid phase extraction (SPE) supported enrichment of LMW proteins, followed by two-dimensional LC-MS top-down analysis encompassing both HCD and EThcD ion activation. Bottom-up experiments were used to support and confirm top-down data interpretation. This strategy allowed for the top-down characterisation 36 proteoforms mapping to 12 SEP of the archaea Methanosarcina mazei, and for the first time the identification of posttranslational modifications in these microproteins.

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Project description:Pattern of gene expression of M. mazei in response to 10 uM bismuth nitrate was analyzed relative to gene expression pattern of M. mazei grown in the presence of 30 uM potassium nitrate (control cultures). Therefore, five pre-cultures of M. mazei was grown to mid-exponential growth-phase. Then, the pre-cultures were divided into aliquotes, one half spiked with bismuth nitrate and the other half with potassium nitrate (control) to match nitrate concentration of the bismuth spike. Samples were propagated for two days. Then, total RNA was isolated and transformed into Cy3 or Cy5 labeled cDNA. Labeled cDNA derived from one culture treated with bismuth and from one control culture, both obtained from the same pre-culture, was hybridized with microarray chips. Hybridized chips were scanned, derived data processed and relative expression levels of orfs from M. mazei in response to bismuth calculated.

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Transcription profiling of Methanosarcina mazei grown with trimethylamine or methanol to investigate the expression pattern of methyltransferase genes

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Project description:The recent discovery of an increasing number of small open reading frame (sORF) creates the need for suitable analytical technologies for the comprehensive identification of the corresponding gene products. In the present study we evaluated analytical approaches which will allow for simultaneous analysis of widest parts of proteome together with the predicted sORF, as for biological investigations and functional studies the knowledge of the entire set of proteins and sORF gene products is essential. We performed a full proteome analysis of the methane producing archeae Methanosarcina mazei cytosolic proteome using a high/low pH reversed phase LC-MS bottom-up approach. The second analytical approach was based on semi-top strategy, encompassing a separation at intact protein level using a GelFree system, followed by digestion and LC-MS analysis. A high overlap in identified proteins was found for both approaches yielding the most comprehensive coverage of the cytosolic proteome of this organism achieved so far. The application of the second approach and an adjustment of the search criteria for database searches further led to a significant increase of sORF peptide identifications, finally allowing to identify 30 sORF gene products.

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Multi-protease approach for the improved identification and molecular characterization of small proteins and short open reading frame-encoded peptides

Project description:The identification of proteins below 70 amino acids in bottom-up proteomics is still a challenging task due to the limited number of peptides generated by proteolytic digestion. This includes the short open reading frame-encoded peptides (SEP), which are a subset of the small proteins that were not previously annotated or that are alternatively encoded. Here, we systematically investigated the use of multiple proteases (trypsin, chymotrypsin, LysC, LysArgiNase and GluC) in GeLC-MS/MS analysis to improve the sequence coverage and the number of identified peptides for small proteins (<70 amino acids), with a focus on SEP, in the archaeon Methanosarcina mazei. Combining the data of all proteases, we identified 63 small proteins and additional 28 SEP with at least two unique peptides, while only 55 small proteins and 22 SEP could be identified using trypsin only. For 27 small proteins and 12 SEP, a 100 % sequence coverage could be achieved. Moreover, for five SEP, incorrectly predicted translation start points were identified, confirming the data of a previous top-down proteomics study of this organism. The results show clearly that a multi-protease approach can improve the identification and molecular characterization of small proteins and SEP.

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