Petri Kouvonen

Novel Proteomics Strategy Brings Insight into the Prevalence of SUMO-2 Target Sites

Small ubiquitin-like modifier (SUMO) is covalently conjugated to its target proteins thereby altering their activity. The mammalian SUMO protein family includes four members (SUMO-1–4) of which SUMO-2 and SUMO-3 are conjugated in a stress-inducible manner. The vast majority of known SUMO substrates are recognized by the single SUMO E2-conjugating enzyme Ubc9 binding to a consensus tetrapeptide (ΨKXE where Ψ stands for a large hydrophobic amino acid) or extended motifs that contain phosphorylated or negatively charged amino acids called PDSM (phosphorylation-dependent sumoylation motif) and NDSM (negatively charged amino acid-dependent sumoylation motif), respectively. We identified 382 SUMO-2 targets using a novel method based on SUMO protease treatment that improves separation of SUMO substrates on SDS-PAGE before LC-ESI-MS/MS. We also implemented a software SUMOFI (SUMO motif finder) to facilitate identification of motifs for SUMO substrates from a user-provided set of proteins and to classify the substrates according to the type of SUMO-targeting consensus site. Surprisingly more than half of the substrates lacked any known consensus site, suggesting that numerous SUMO substrates are recognized by a yet unknown consensus site-independent mechanism. Gene ontology analysis revealed that substrates in distinct functional categories display strikingly different prevalences of NDSM sites. Given that different types of motifs are bound by Ubc9 using alternative mechanisms, our data suggest that the preference of SUMO-2 targeting mechanism depends on the biological function of the substrate.

Microarray profiling of host-extract-induced genes and characterization of the type VI secretion cluster in the potato pathogen Pectobacterium atrosepticum.

Pectobacterium atrosepticum is a Gram-negative plant-pathogenic bacterium that rots potato stems and tubers. Microarray analysis was used to identify genes that were differentially expressed when host extracts were added to the growth medium. Potato extracts downregulated the expression of ribosomal genes and genes related to uptake and metabolism of nutrients, and upregulated genes needed for nitrate or phosphonate use. Some of the observed changes in gene expression in host-extract-induced cultures are similar to those during attachment of the bacterium to host tissues. Other responses indicated defence against toxic metabolites in the extract. Tuber extract induced a large gene cluster having homology to type VI secretion genes shown to be virulence determinants in many, but not all, animal and human pathogens. Two of the genes in the type VI cluster were found to be expressed during infection in potato tubers and stems, and mutants with knockouts of the corresponding genes had increased virulence on potato. One of the type VI secretion mutants was further characterized and found to grow to higher cell density in culture in the presence of host extract and to produce slightly more extracellular tissue-macerating enzymes than the wild-type strain. Analysis of secreted proteins showed that this type VI mutant was affected in the production of haemolysin-coregulated proteins (Hcps), which have been suggested to be secreted by the type VI pathway in other bacteria. The results suggest that the type VI secretion system of P. atrosepticum is needed for secretion of Hcps but not for virulence on its host plant, potato.

Quantitative proteomics reveals GIMAP family proteins 1 and 4 to be differentially regulated during human T helper cell differentiation

T helper (Th) cells differentiate into functionally distinct effector cell subsets of which Th1 and Th2 cells are best characterized. Besides T cell receptor signaling, IL-12-induced STAT4 and T-bet- and IL-4-induced STAT6 and GATA3 signaling pathways are the major players regulating the Th1 and Th2 differentiation process, respectively. However, there are likely to be other yet unknown factors or pathways involved. In this study we used quantitative proteomics exploiting cleavable ICAT labeling and LC-MS/MS to identify IL-4-regulated proteins from the microsomal fractions of CD4+ cells extracted from umbilical cord blood. We were able to identify 557 proteins of which 304 were also quantified. This study resulted in the identification of the down-regulation of small GTPases GIMAP1 and GIMAP4 by IL-4 during Th2 differentiation. We also showed that both GIMAP1 and GIMAP4 genes are up-regulated by IL-12 and other Th1 differentiation-inducing cytokines in cells induced to differentiate toward Th1 lineage and down-regulated by IL-4 in cells induced to Th2. Our results indicate that the GIMAP (GTPase of the immunity-associated protein) family of proteins is differentially regulated during Th cell differentiation.

Development of a Quantitative SRM-Based Proteomics Method to Study Iron Metabolism of Synechocystis sp. PCC 6803

The cyanobacterium Synechocystis sp. PCC 6803 (S. 6803) is a well-established model species in oxygenic photosynthesis research and a potential host for biotechnological applications. Despite recent advances in genome sequencing and microarray techniques applied in systems biology, quantitative proteomics approaches with corresponding accuracy and depth are scarce for S. 6803. In this study, we developed a protocol to screen changes in the expression of 106 proteins representing central metabolic pathways in S. 6803 with a targeted mass spectrometry method, selected reaction monitoring (SRM). We evaluated the response to the exposure of both short- and long-term iron deprivation. The experimental setup enabled the relative quantification of 96 proteins, with 87 and 92 proteins showing adjusted p-values <0.01 under short- and long-term iron deficiency, respectively. The high sensitivity of the SRM method for S. 6803 was demonstrated by providing quantitative data for altogether 64 proteins that previously could not be detected with the classical data-dependent MS approach under similar conditions. This highlights the effectiveness of SRM for quantification and extends the analytical capability to low-abundance proteins in unfractionated samples of S. 6803. The SRM assays and other generated information are now publicly available via PASSEL and Panorama.

Identification of differentially expressed proteins in Ficedula flycatchers

This is the first report on the large‐scale identification and comparison of proteins in non‐model organisms, Ficedula flycatchers. It highlights the potential of proteomics approaches in both non‐sequenced and non‐model organisms for identification of differentially expressed proteins. Not surprisingly, more than 55% of the proteins failed to be identified even though the MS spectra were of high quality. Nevertheless, the protein information obtained in this study will serve as a valuable resource for continued research.

On-target ultrasonic digestion of proteins

In the present work, we report a novel on‐target protein cleavage method. The method utilizes ultrasonic energy and allows up to 20 samples to be cleaved in 5 min for protein identification and one sample in 30 s for on‐tissue digestion. The standard proteins were spotted on a conductive glass slide in a volume of 0.5 μL followed by 5 min of ultrasonication after trypsin addition. Controls (5 min, 37°C no ultrasonication) were also assayed. After trypsin addition, digestion of the tissues was enhanced by 30 s of ultrasonication. The samples were analyzed and compared to those obtained by using conventional 3 h heating proteolysis. The low sample volume needed for the digestion and reduction in sample‐handling steps and time are the features that make this method appealing to the many laboratories working with high‐throughput sample treatment.

Isolation and identification of cyclic lipopeptides from Paenibacillus ehimensis, strain IB-X-b.

Antifungal lipopeptides produced by an antagonistic bacterium, Paenibacillus ehimensis strain IB-X-b, were purified and analyzed. The acetone extract of the culture supernatant contained an antifungal amphiphilic fraction stainable with ninhydrin on thin layer chromatography. The fraction was further purified with water-methanol extraction followed by a chromatography on a C18-support. The analysis with LC-MS showed presence of two main series of homologous compounds, family of depsipeptides containing a hydroxy fatty acid, three 2,4-diaminobutyric acid (Dab) residues, five hydrophobic amino acids and one Ser/Thr residue, and cyclic lipopeptides of bacillomycin L and fengycin/plipastatin/agrastatin families. The prevailing compounds in this group are bacillomycin L-C15, fengycin/plipastatin A-C16 together with their homologues responsible for the majority of fungal growth inhibition by P. ehimensis IB-X-b.

Isotopic labelling of peptides in tissues enhances mass spectrometric profiling.

RATIONALE There is a need in imaging mass spectrometry to use the acquired isotope distribution to unequivocally determine the identity of a peptide ion. A way of achieving unambiguous differentiation of ions from protonated peptides from other [M + H](+) ions in a tissue would be via the direct on-tissue incorporation of (18)O into peptides. METHODS Tissues were first digested with trypsin for 3  h at 37 °C in a humidified chamber. For the (18)O-labelling of digested peptides 1  μL of H(2)(18)O/50  mM ammonium acetate (at pH 6.75) was added to the array of tryptic spots and incubated at room temperature for 20  min. α-Cyano-4-hydroxycinnamic acid was used as a matrix modifier. The mass spectral analysis of tissue sections was carried out using a matrix-assisted laser desorption/ionisation tandem time-of-flight (MALDI-TOF-TOF) instrument. RESULTS On-tissue incorporation of (18)O into peptides cannot be carried out during the digestion step inside a humidified chamber. After tissue digestion for 3  h at 37 °C in an humidified chamber, (18)O labelling was carried out for 20  min at room temperature (no humidified chamber). No trypsin was needed to enhance the labelling. CONCLUSIONS For first time the feasibility of (18)O-labelling of peptides in situ for tissues has been demonstrated. The method decouples protein digestion from peptide labelling and is performed in sequential steps. Furthermore, we observed that (18)O incorporation produces characteristic isotopic peptide distributions, thus making facile distinguishing peptides from other tissue molecular components that ionise in the MALDI ion source.

Nitromatrix provides improved LC-MALDI signals and more protein identifications

The beneficial use of NC in MALDI‐MS has previously been reported to provide better S/N and reproducibility as well as less alkali metal adducts. We have therefore investigated if additional beneficial properties of NC also existed for commonly employed proteomics‐based LC‐MALDI procedures. Specifically we studied the effects of NC as a matrix cofactor for prestructured sample supports (AnchorChip plates), and compared the performance with several alternative sample preparation methods recently reported in the literature. The work reported here describes a new method of mixing the NC‐matrix solution with the LC‐eluent prior to sample deposition and shows that a mixture of CHCA and NC in a complex solvent offers superior analytical results in several ways: most striking is the higher signal intensity, and that the signals last much longer, due to the robustness of the matrix formulation. We have tested the use of the nitromatrix on a single LC‐MALDI preparation and found that at least ten reiterative analyses could be performed, resulting in total analysis times of more than 75 h (approximately 15 million laser shots). Consequently more than twice as many proteins could be identified than from a single analysis. This combination of longer, and stronger, MALDI signals provided an increase in the number of peptides, greater sequence coverage in MS/MS experiments and ultimately more confident peptide assignments.

A journey through PROTEOSONICS.

Ultrasonic energy is gaining momentum in Proteomics. It helps to shorten many proteomics workflows in an easy and efficient manner. Ultrasonic energy is nowadays used for protein extraction, solubilisation and cell disruption, to speed protein identification, protein quantification, peptide profiling, metal-protein complexes characterisation and imaging mass spectrometry. The present review gives a perspective of the latest achievements in ultrasonic-based sample treatment for proteomics as well as provides the basic concepts and the tools of the trade to efficiently implement this tool in proteomics labs.