Publications by authors named "Michael Stadlmeier"

6 Publications

  • Page 1 of 1

TMTpro Complementary Ion Quantification Increases Plexing and Sensitivity for Accurate Multiplexed Proteomics at the MS2 Level.

J Proteome Res 2021 Apr 30. Epub 2021 Apr 30.

Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, United States.

Multiplexed proteomics is a powerful tool to assay cell states in health and disease, but accurate quantification of relative protein changes is impaired by interference from co-isolated peptides. Interference can be reduced by using MS3-based quantification, but this reduces sensitivity and requires specialized instrumentation. An alternative approach is quantification by complementary ions, the balancer group-peptide conjugates, which allows accurate and precise multiplexed quantification at the MS2 level and is compatible with most proteomics instruments. However, complementary ions of the popular TMT-tag form inefficiently and multiplexing is limited to five channels. Here, we evaluate and optimize complementary ion quantification for the recently released TMTpro-tag, which increases complementary ion plexing capacity to eight channels (TMTproC). Furthermore, the beneficial fragmentation properties of TMTpro increase sensitivity for TMTproC, resulting in ∼65% more proteins quantified compared to TMTpro-MS3 and ∼18% more when compared to real-time-search TMTpro-MS3 (RTS-SPS-MS3). TMTproC quantification is more accurate than TMTpro-MS2 and even superior to RTS-SPS-MS3. We provide the software for quantifying TMTproC data as an executable that is compatible with the MaxQuant analysis pipeline. Thus, TMTproC advances multiplexed proteomics data quality and widens access to accurate multiplexed proteomics beyond laboratories with MS3-capable instrumentation.
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http://dx.doi.org/10.1021/acs.jproteome.0c00813DOI Listing
April 2021

The cGMP-Dependent Protein Kinase 2 Contributes to Cone Photoreceptor Degeneration in the -Deficient Mouse Model of Achromatopsia.

Int J Mol Sci 2020 Dec 23;22(1). Epub 2020 Dec 23.

Department of Pharmacy-Center for Drug Research, Ludwig-Maximilians-University, 81377 Munich, Germany.

Mutations in the gene, which encodes the A subunit of the cyclic guanosine monophosphate (cGMP)-gated cation channel in cone photoreceptor outer segments, cause total colour blindness, also referred to as achromatopsia. Cones lacking this channel protein are non-functional, accumulate high levels of the second messenger cGMP and degenerate over time after induction of ER stress. The cell death mechanisms that lead to loss of affected cones are only partially understood. Here, we explored the disease mechanisms in the knockout (KO) mouse model of achromatopsia. We found that another important effector of cGMP, the cGMP-dependent protein kinase 2 (Prkg2) is crucially involved in cGMP cytotoxicity of cones in KO mice. Virus-mediated knockdown or genetic ablation of in KO mice counteracted degeneration and preserved the number of cones. Analysis of markers of endoplasmic reticulum stress and unfolded protein response confirmed that induction of these processes in KO cones also depends on Prkg2. In conclusion, we identified Prkg2 as a novel key mediator of cone photoreceptor degeneration in achromatopsia. Our data suggest that this cGMP mediator could be a novel pharmacological target for future neuroprotective therapies.
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http://dx.doi.org/10.3390/ijms22010052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7793084PMC
December 2020

A Click-Chemistry-Based Enrichable Crosslinker for Structural and Protein Interaction Analysis by Mass Spectrometry.

Chembiochem 2020 01 28;21(1-2):103-107. Epub 2019 Nov 28.

Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstrasse 5-13, 81377, Munich, Germany.

Mass spectrometry is the method of choice for the characterisation of proteomes. Most proteins operate in protein complexes, in which their close association modulates their function. However, with standard MS analysis, information on protein-protein interactions is lost and no structural information is retained. To gain structural and interactome data, new crosslinking reagents are needed that freeze inter- and intramolecular interactions. Herein, the development of a new reagent, which has several features that enable highly sensitive crosslinking MS, is reported. The reagent enables enrichment of crosslinked peptides from the majority of background peptides to facilitate efficient detection of low-abundant crosslinked peptides. Due to the special cleavable properties, the reagent can be used for MS and potentially for MS experiments. Thus, the new crosslinking reagent, in combination with high-end MS, should enable sensitive analysis of interactomes, which will help researchers to obtain important insights into cellular states in health and diseases.
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http://dx.doi.org/10.1002/cbic.201900611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980279PMC
January 2020

Noncanonical RNA Nucleosides as Molecular Fossils of an Early Earth-Generation by Prebiotic Methylations and Carbamoylations.

Angew Chem Int Ed Engl 2018 05 17;57(20):5943-5946. Epub 2018 Apr 17.

Center for Integrated Protein Science (CiPSM) at the Department, of Chemistry, LMU München, Butenandtstrasse 5-13, 81377, München, Germany.

The RNA-world hypothesis assumes that life on Earth started with small RNA molecules that catalyzed their own formation. Vital to this hypothesis is the need for prebiotic routes towards RNA. Contemporary RNA, however, is not only constructed from the four canonical nucleobases (A, C, G, and U), it also contains many chemically modified (noncanonical) bases. A still open question is whether these noncanonical bases were formed in parallel to the canonical bases (chemical origin) or later, when life demanded higher functional diversity (biological origin). Here we show that isocyanates in combination with sodium nitrite establish methylating and carbamoylating reactivity compatible with early Earth conditions. These reactions lead to the formation of methylated and amino acid modified nucleosides that are still extant. Our data provide a plausible scenario for the chemical origin of certain noncanonical bases, which suggests that they are fossils of an early Earth.
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http://dx.doi.org/10.1002/anie.201801919DOI Listing
May 2018

A Sulfoxide-Based Isobaric Labelling Reagent for Accurate Quantitative Mass Spectrometry.

Angew Chem Int Ed Engl 2018 03 15;57(11):2958-2962. Epub 2018 Feb 15.

Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377, Munich, Germany.

Modern proteomics requires reagents for exact quantification of peptides in complex mixtures. Peptide labelling is most typically achieved with isobaric tags that consist of a balancer and a reporter part that separate in the gas phase. An ingenious distribution of stable isotopes provides multiple reagents with identical molecular weight but a different mass of the reporter groups, allowing relative quantification of multiple samples in one measurement. Here we report a new isobaric labelling reagent, where the balancer and the reporter are linked by a sulfoxide group, which, based on the sulfoxide pyrolysis, leads to easy and asymmetric cleavage at low fragmentation energy. The fragmentation of our new design is significantly improved, yielding more intense complementary ion signals, allowing complementary ion cluster analysis as well.
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http://dx.doi.org/10.1002/anie.201708867DOI Listing
March 2018

Orchestrating the biosynthesis of an unnatural pyrrolysine amino Acid for its direct incorporation into proteins inside living cells.

Chemistry 2015 May 2;21(21):7701-4. Epub 2015 Apr 2.

Center for Integrative Protein Science at the Department of Chemistry, Ludwig-Maximilians Universität München, Butenandtstrasse 5-13, 81377 Munich (Germany).

We here report the construction of an E. coli expression system able to manufacture an unnatural amino acid by artificial biosynthesis. This can be orchestrated with incorporation into protein by amber stop codon suppression inside a living cell. In our case an alkyne-bearing pyrrolysine amino acid was biosynthesized and incorporated site-specifically allowing orthogonal double protein labeling.
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http://dx.doi.org/10.1002/chem.201500971DOI Listing
May 2015