Next-generation protein analysis in the pathology department

Traditionally, immunohistochemistry (IHC) is used by pathologists to localise specific proteins or peptides in tissue slides. In the era of personalised medicine, however, molecular tissue analysis becomes indispensable for correct diagnosis, prognosis and therapeutic decision, not only on the DNA or mRNA level but also on the protein level. Combining molecular information with imaging presents many advantages. Therefore, matrix-assisted laser desorption/ionisation imaging mass spectrometry (MALDI IMS) is a promising technique to be added to the armamentarium of the pathologist. Here, we focus on the workflow, advantages and drawbacks of both MALDI IMS and IHC. We also briefly discuss a few other protein imaging modalities and give examples of applications

PPE38-Secretion-Dependent Proteins of M. tuberculosis Alter NF-kB Signalling and Inflammatory Responses in Macrophages

It was previously shown that secretion of PE-PGRS and PPE-MPTR proteins is abolished in clinical M. tuberculosis isolates with a deletion in the ppe38-71 operon, which is associated with increased virulence. Here we investigate the proteins dependent on PPE38 for their secretion and their role in the innate immune response using temporal proteomics and protein turnover analysis in a macrophage infection model. A decreased pro-inflammatory response was observed in macrophages infected with PPE38-deficient M. tuberculosis CDC1551 as compared to wild type bacteria. We could show that dampening of the pro-inflammatory response is associated with activation of a RelB/p50 pathway, while the canonical inflammatory pathway is active during infection with wild type M. tuberculosis CDC1551. These results indicate a molecular mechanism by which M. tuberculosis PE/PPE proteins controlled by PPE38 have an effect on modulating macrophage responses through NF-kB signalling

Next-generation protein analysis in the pathology department

Traditionally, immunohistochemistry (IHC) is used by pathologists to localise specific proteins or peptides in tissue slides. In the era of personalised medicine, however, molecular tissue analysis becomes indispensable for correct diagnosis, prognosis and therapeutic decision, not only on the DNA or mRNA level but also on the protein level. Combining molecular information with imaging presents many advantages. Therefore, matrix-assisted laser desorption/ionisation imaging mass spectrometry (MALDI IMS) is a promising technique to be added to the armamentarium of the pathologist. Here, we focus on the workflow, advantages and drawbacks of both MALDI IMS and IHC. We also briefly discuss a few other protein imaging modalities and give examples of applications

PPE38-Secretion-Dependent Proteins of M. tuberculosis Alter NF-kB Signalling and Inflammatory Responses in Macrophages

It was previously shown that secretion of PE-PGRS and PPE-MPTR proteins is abolished in clinical M. tuberculosis isolates with a deletion in the ppe38-71 operon, which is associated with increased virulence. Here we investigate the proteins dependent on PPE38 for their secretion and their role in the innate immune response using temporal proteomics and protein turnover analysis in a macrophage infection model. A decreased pro-inflammatory response was observed in macrophages infected with PPE38-deficient M. tuberculosis CDC1551 as compared to wild type bacteria. We could show that dampening of the pro-inflammatory response is associated with activation of a RelB/p50 pathway, while the canonical inflammatory pathway is active during infection with wild type M. tuberculosis CDC1551. These results indicate a molecular mechanism by which M. tuberculosis PE/PPE proteins controlled by PPE38 have an effect on modulating macrophage responses through NF-kB signalling

Multiple solvent elution, a method to counter the effects of coelution and ion suppression in LC-MS analysis in bottom up proteomics

On average a human cell type expresses around 10,000 different protein coding genes synthesizing all the different molecular forms of the protein product (proteoforms) found in a cell. In a typical shotgun bottom up proteomic approach, the proteins are enzymatically cleaved, producing several 100,000 s of different peptides that are analyzed with liquid chromatography-tandem mass spectrometry (LC-MSMS). One of the major consequences of this high sample complexity is that coelution of peptides cannot be avoided. Moreover, low abundant peptides are difficult to identify as they have a lower chance of being selected for fragmentation due to ion-suppression effects and the semi-stochastic nature of the precursor selection in data-dependent shotgun proteomic analysis where peptides are selected for fragmentation analysis one-by-one as they elute from the column. In the current study we explore a simple novel approach that has the potential to counter some of the effect of coelution of peptides and improves the number of peptide identifications in a bottom-up proteomic analysis. In this method, peptides from a HeLa cell digest were eluted from the reverse phase column using three different elution solvents (acetonitrile, methanol and acetone) in three replicate reversed phase LC-MS/MS shotgun proteomic analysis. Results were compared with three technical replicates using the same solvent, which is common practice in proteomic analysis. In total, we see an increase of up to 10% in unique protein and up to 30% in unique peptide identifications from the combined analysis using different elution solvents when compared to the combined identifications from the three replicates of the same solvent. In addition, the overlap of unique peptide identifications common in all three LC-MS analyses in our approach is only 23% compared to 50% in the replicates using the same solvent. The method presented here thus provides an easy to implement method to significantly reduce the effects of coelution and ion suppression of peptides and improve protein coverage in shotgun proteomics. Data are available via ProteomeXchange with identifier PXDO11908

Multiple solvent elution, a method to counter the effects of coelution and ion suppression in LC-MS analysis in bottom up proteomics

On average a human cell type expresses around 10,000 different protein coding genes synthesizing all the different molecular forms of the protein product (proteoforms) found in a cell. In a typical shotgun bottom up proteomic approach, the proteins are enzymatically cleaved, producing several 100,000 s of different peptides that are analyzed with liquid chromatography-tandem mass spectrometry (LC-MSMS). One of the major consequences of this high sample complexity is that coelution of peptides cannot be avoided. Moreover, low abundant peptides are difficult to identify as they have a lower chance of being selected for fragmentation due to ion-suppression effects and the semi-stochastic nature of the precursor selection in data-dependent shotgun proteomic analysis where peptides are selected for fragmentation analysis one-by-one as they elute from the column. In the current study we explore a simple novel approach that has the potential to counter some of the effect of coelution of peptides and improves the number of peptide identifications in a bottom-up proteomic analysis. In this method, peptides from a HeLa cell digest were eluted from the reverse phase column using three different elution solvents (acetonitrile, methanol and acetone) in three replicate reversed phase LC-MS/MS shotgun proteomic analysis. Results were compared with three technical replicates using the same solvent, which is common practice in proteomic analysis. In total, we see an increase of up to 10% in unique protein and up to 30% in unique peptide identifications from the combined analysis using different elution solvents when compared to the combined identifications from the three replicates of the same solvent. In addition, the overlap of unique peptide identifications common in all three LC-MS analyses in our approach is only 23% compared to 50% in the replicates using the same solvent. The method presented here thus provides an easy to implement method to significantly reduce the effects of coelution and ion suppression of peptides and improve protein coverage in shotgun proteomics. Data are available via ProteomeXchange with identifier PXDO11908