Comprehensive analysis of post-translational modifications and protein variants
The facility employs advanced liquid chromatography - mass spectrometry (LC-MS/MS) to answer diverse biological questions. We are specialized in quantitative analyses of different protein forms, and often combine affinity enrichment protocols with mass spectrometry readout to quantify specific groups of proteins or peptides. We routinely work with different types of biological samples, from cell lines to tissues, as well as do analysis of proteins from different compartments.
Phosphorylation, methylation and glycosylation are post-translational modifications for which we have optimised protocols, and for which we continuously further develop sample processing protocols. We assist with proteome analyses using all common quantitative approaches, such as label-free, SILAC and mass tag (TMT)-based quantification.
- Human cell line / tissue - fast quantitative proteome (4000-5000 proteins/sample in 2-3 hours) for relative comparisons of proteomes;
- Human plasma – fast quantitative proteome (300-400 proteins / sample in 2-3 hours). Tens to hundreds of samples;
- PTM proteomics - quantitative analyses of phosphorylation, methylation, acetylation etc.
- Interactomics – analysis of protein complexes and interactions in cells;
- Deep proteome analyses – identification of up to 10.000 proteins in complex mammalian proteomes such as cell line or tissue;
- Immunoprecipitation assays for the identification of antibody-antigen interactions;
- Secretome or specific organelle proteome analysis;
- Single-cell proteomics.
The facility operates a QExactive HF-X mass spectrometer which is operated in-line with an Easy nano-LC system for peptide separation. This combination allows for quantification of about 5000 proteins in mammalian cell samples in two-hour time, without sample pre-fractionation. We also have an EvoSep One LC system that allows for high-throughput analyses.
The QE HF-X is a Hybrid Quadrupole Orbitrap with great sensitivity and resolution, allowing for quantification of peptides in complex peptide mixtures in explorative, qualitative and quantitative proteomic studies. Furthermore, the instrument allows targeted analysis of specific peptides and proteins of interest through parallel reaction monitoring (PRM).
Example publications from Proteoforms@LU usage:
- HC Tran et al. An mTRAN-mRNA interaction mediates mitochondrial translation initiation in plants (2023) Science. https://doi.org/10.1126/science.adg0995
- S Mosquim Junior et al Choice of High-Throughput Proteomics Method Affects Data Integration with Transcriptomics and the Potential Use in Biomarker Discovery (2022) Cancers https://doi.org/10.3390/cancers14235761
- V Siino et al Plasma proteome profiling of healthy individuals across the life span in a Sicilian cohort with long-lived individuals (2022) Aging Cell https://doi.org/10.1111/acel.13684
- C Robertsson et al Proteomic response in Streptococcus gordonii DL1 biofilm cells during attachment to salivary MUC5B (2021) J Oral Microbiol https://doi.org/10.1080/20002297.2021.1967636
- E Davydova et al The methyltransferase METTL9 mediates pervasive 1-methylhistidine modification in mammalian proteomes (2021) Nat Communhttps://doi.org/10.1038/s41467-020-20670-7