Phagosome proteomics: a powerful tool to assess bacteria-mediated immunomodulation

Bacteria-mediated immunomodulation has important implications in microbial infection and bacterial vaccines. Intraphagosomal bacteria negotiate survival niches with the intracellular environment by modulating the phagosome composition during invasion. The final phagosome composition determines the fate of the intraphagosomal bacterium or the efficacy of a bacterial vaccine. Therefore, the phagosome proteome is a valuable readout to assess the ability of a natural or genetically engineered bacterial strain to modulate the host immune response. Compared to a preparation of latex-bead-containing phagosomes, the preparation of bacterial phagosomes requires additional measures to ensure comparable purity due to their closer density to some other organelles. This bottleneck can be overcome with delicate preparation protocols, proper experimental designs to facilitate bioinformatics based discrimination against contaminating proteins, and the incorporation of stable-isotope labeled internal standards to correct for contaminating fractions of phagosomal proteins. The rapid progress in the proteomics and bioinformatics fields provides an array of techniques that promise to bring about an unprecedented coverage of both known and as yet undiscovered immunomodulation pathways within bacterial phagosome proteomes. A precise portrait of the bacteria-mediated immunomodulation pathways in phagosomes will likely aid in the intelligent design of bioengineered bacterial vaccine strains for important future biomedical applications.     

A Solanum neorickii introgression population providing a powerful complement to the extensively characterized Solanum pennellii population

We present a complementary resource for trait fine‐mapping in tomato to those based on the intra‐specific cross between cultivated tomato and the wild tomato species Solanum pennellii, which have been extensively used for quantitative genetics in tomato over the last 20 years. The current population of backcross inbred lines (BILs) is composed of 107 lines derived after three backcrosses of progeny of the wild species Solanum neorickii (LA2133) and cultivated tomato (cultivar TA209) and is freely available to the scientific community. These S. neorickii BILs were genotyped using the 10K SolCAP single nucleotide polymorphism chip, and 3111 polymorphic markers were used to map recombination break points relative to the physical map of Solanum lycopersicum. The BILs harbor on average 4.3 introgressions per line, with a mean introgression length of 34.7 Mbp, allowing partitioning of the genome into 340 bins and thereby facilitating rapid trait mapping. We demonstrate the power of using this resource in comparison with archival data from the S. pennellii resources by carrying out metabolic quantitative trait locus analysis following gas chromatography–mass spectrometry on fruits harvested from the S. neorickii BILs. The metabolic candidate genes phenylalanine ammonia‐lyase and cystathionine gamma‐lyase were then tested and validated in F₂ populations and via agroinfiltration‐based overexpression in order to exemplify the fidelity of this method in identifying the genes that drive tomato metabolic phenotypes.     

A label-free quantification method by MS/MS TIC compared to SILAC and spectral counting in a proteomics screen

In order to assess the biological function of proteins and their modifications for understanding signaling mechanisms within cells as well as specific biomarkers to disease, it is important that quantitative information be obtained under different experimental conditions. Stable isotope labeling is a powerful method for accurately determining changes in the levels of proteins and PTMs; however, isotope labeling experiments suffer from limited dynamic range resulting in signal change ratios of less than approximately 20:1 using most commercial mass spectrometers. Label-free approaches to relative quantification in proteomics such as spectral counting have gained popularity since no additional chemistries are needed. Here, we show a label-free method for relative quantification based on the TIC from peptide MS/MS spectra collected from data-dependent runs can be used effectively as a quantitative measure and expands the dynamic range over isotope labeling experiments allowing for abundance differences up to approximately 60:1 in a screen for proteins that bind to phosphotyrosine residues.     

The importance of the digest: proteolysis and absolute quantification in proteomics

Virtually all mass spectrometric-based methods for quantitative proteomics are at the peptide level, whether label-mediated or label-free. Absolute quantification in particular is based on the measurement of limit peptides, defined as those peptides that cannot be further fragmented by the protease in use. Complete release of analyte and (stable isotope labelled) standard ensures that the most reliable quantification data are recovered, especially when the standard peptides are in a different primary sequence context, such as sometimes occurs in the QconCAT methodology. Moreover, in label-free methods, incomplete digestion would diminish the ion current attributable to limit peptides and lead to artifactually low quantification data. It follows that an essential requirement for peptide-based absolute quantification in proteomics is complete and consistent proteolysis to limit peptides. In this paper we describe strategies to assess completeness of proteolysis and discuss the potential for variance in digestion efficiency to compromise the ensuing quantification data. We examine the potential for kinetically favoured routes of proteolysis, particularly at the last stages of the digestion, to direct products into ‘dead-end’ mis-cleaved products.     

Unique scanning capabilities of a new hybrid linear ion trap mass spectrometer (Q TRAP) used for high sensitivity proteomics applications

The unique scanning capabilities of a hybrid linear ion trap (Q TRAP) mass spectrometer are described with an emphasis on proteomics applications. The combination of the very selective triple quadrupole based tandem mass spectrometry (MS/MS) scans with the very sensitive ion trap product ion scans allows rapid identification of peptides at low concentrations derived from post-translationally modified proteins on chromatographic time scales. The Q TRAP instrument also offers the opportunity to conduct a variety of ion processing steps prior to performing a mass scan. For example, the enhancement of the multiple-charge ion contents of the ion trap can be performed resulting in a survey mass spectrum dominated by double- and triple-charge peptides. This facilitates the identification of relevant biological species in both separated and unseparated peptide mixtures for further MS/MS experiments.     

Top-down quantitative proteomics identified phosphorylation of cardiac troponin I as a candidate biomarker for chronic heart failure

The rapid increase in the prevalence of chronic heart failure (CHF) worldwide underscores an urgent need to identify biomarkers for the early detection of CHF. Post-translational modifications (PTMs) are associated with many critical signaling events during disease progression and thus offer a plethora of candidate biomarkers. We have employed a top-down quantitative proteomics methodology for comprehensive assessment of PTMs in whole proteins extracted from normal and diseased tissues. We systematically analyzed 36 clinical human heart tissue samples and identified phosphorylation of cardiac troponin I (cTnI) as a candidate biomarker for CHF. The relative percentages of the total phosphorylated cTnI forms over the entire cTnI populations (%P(total)) were 56.4 ± 3.5%, 36.9 ± 1.6%, 6.1 ± 2.4%, and 1.0 ± 0.6% for postmortem hearts with normal cardiac function (n = 7), early stage of mild hypertrophy (n = 5), severe hypertrophy/dilation (n = 4), and end-stage CHF (n = 6), respectively. In fresh transplant samples, the %P(total) of cTnI from nonfailing donor (n = 4), and end-stage failing hearts (n = 10) were 49.5 ± 5.9% and 18.8 ± 2.9%, respectively. Top-down MS with electron capture dissociation unequivocally localized the altered phosphorylation sites to Ser22/23 and determined the order of phosphorylation/dephosphorylation. This study represents the first clinical application of top-down MS-based quantitative proteomics for biomarker discovery from tissues, highlighting the potential of PTMs as disease biomarkers.     

The importance of peptide detectability for protein identification, quantification, and experiment design in MS/MS proteomics

Peptide detectability is defined as the probability that a peptide is identified in an LC-MS/MS experiment and has been useful in providing solutions to protein inference and label-free quantification. Previously, predictors for peptide detectability trained on standard or complex samples were proposed. Although the models trained on complex samples may benefit from the large training data sets, it is unclear to what extent they are affected by the unequal abundances of identified proteins. To address this challenge and improve detectability prediction, we present a new algorithm for the iterative learning of peptide detectability from complex mixtures. We provide evidence that the new method approximates detectability with useful accuracy and, based on its design, can be used to interpret the outcome of other learning strategies. We studied the properties of peptides from the bacterium Deinococcus radiodurans and found that at standard quantities, its tryptic peptides can be roughly classified as either detectable or undetectable, with a relatively small fraction having medium detectability. We extend the concept of detectability from peptides to proteins and apply the model to predict the behavior of a replicate LC-MS/MS experiment from a single analysis. Finally, our study summarizes a theoretical framework for peptide/protein identification and label-free quantification.     

Top-down protein sequencing and MS3 on a hybrid linear quadrupole ion trap-orbitrap mass spectrometer

Top-down proteomics, the analysis of intact proteins (instead of first digesting them to peptides), has the potential to become a powerful tool for mass spectrometric protein characterization. Requirements for extremely high mass resolution, accuracy, and ability to efficiently fragment large ions have often limited top-down analyses to custom built FT-ICR mass analyzers. Here we explore the hybrid linear ion trap (LTQ)-Orbitrap, a novel, high performance, and compact mass spectrometric analyzer, for top-down proteomics. Protein standards from 10 to 25 kDa were electrosprayed into the instrument using a nanoelectrospray chip. Resolving power of 60,000 was ample for isotope resolution of all protein charge states. We achieved absolute mass accuracies for intact proteins between 0.92 and 2.8 ppm using the "lock mass" mode of operation. Fifty femtomole of cytochrome c applied to the chip resulted in spectra with excellent signal-to-noise ratio and only low attomole sample consumption. Different protein charge states were dissociated in the LTQ, and the sensitivity of the orbitrap allowed routine, high resolution, and high mass accuracy fragment detection. This resulted in unambiguous charge state determination of fragment ions and identification of unmodified and modified proteins by database searching. Protein fragments were further isolated and fragmented in the LTQ followed by analysis of MS(3) fragments in the orbitrap, localizing modifications to part of the sequence and helping to identify the protein with these small peptide-like fragments. Given the ready availability and ease of operation of the LTQ-Orbitrap, it may have significant impact on top-down proteomics.     

Development of a high throughput screening method to test flavour-forming capabilities of anaerobic micro-organisms

AIM: Development of a fast, automated and reliable screening method for screening of large collections of bacterial strains with minimal handling time. METHODS AND RESULTS: The method is based on the injection of a small headspace sample (100 microl) from culture vials (2 ml) in 96-well format directly into the mass spectrometry (MS). A special sample tray has been developed for liquid media, and anaerobically grown cultures. In principle, all volatile components can be measured, but a representative mass fragment has to be obtained in the MS. Representative masses for 3-methylbutanal, 2-methylpropanal and benzaldehyde are 58, 72 and 105, respectively. In 1 day over 1500 samples could be analysed and the coefficient of variation for the response was <5%. CONCLUSION: Screening of 72 strains belonging to the genus Lactococcus in quadruple on the production of the key-flavour compound 3-methylbutanal illustrated the effectiveness of the method. Furthermore, knowledge of the biochemistry and physiology of 3-methylbutanal formation was used to optimize the composition of the growth medium to enhance 3-methylbutanal production, and thereby improve the screening. SIGNIFICANCE AND IMPACT OF THE STUDY: A commonly used method to control flavour formation in fermented food products is the selection of bacterial strains, which are able to produce the desired flavour compounds. As large collections of strains are available for such screenings, studying biodiversity of micro-organisms on the level of metabolic routes is strongly facilitated by highly automated high throughput screening methods for measuring enzyme activities or production of metabolites. Therefore, this method will be a useful tool for selecting flavour-producing strains and for enhancing starter culture development.     

Limited proteolysis of a chemically modified third component of human complement, C3, by cathepsin G of human leukocytes

We have investigated the limited proteolysis of the third component of complement, C3, by a human leukocyte protease, cathepsin G, by using a chemically modified C3, which was prepared by treatment of C3 with methylamine and a fluorescent thiol reagent, N-(dimethylamino-4-methylcoumarinyl)-maleimide (DACM) and was thus named DACM-C3me. Although native C3 was hardly cleaved by cathepsin G, DACM-C3me was cleaved by cathepsin G into three major fragments, which were termed C3c-G (150,000 daltons, 150 kd), C3d-G (25 kd), and C3a-G (10 kd). C3c-G was composed of four disulfide-linked polypeptide chains of 75 kd, 35 kd, and two 25 kd. C3d-G and C3a-G were single-chain fragments derived from the alpha chain. The N-terminal sequence of C3d-G was determined as Thr-Glu-Asp-Ala-Val-, suggesting that cathepsin G released C3d-G by cleaving a Met-Thr peptide bond which is located at 19 residues toward the N-terminal from the cysteinyl residue forming an internal thiolester linkage in native C3. C3d-G, like C3d-K (a C3d fragment produced by the action of plasma kallikrein), was found to have bioactivities such as leukocytosis-inducing and immunosuppressive activities.     

Advances in human proteomics at high scale with the SOMAscan proteomics platform

In 1997, while still working at NeXstar Pharmaceuticals, several of us made a proteomic bet. We thought then, and continue to think, that proteomics offers a chance to identify disease-specific biomarkers and improve healthcare. However, interrogating proteins turned out to be a much harder problem than interrogating nucleic acids. Consequently, the 'omics' revolution has been fueled largely by genomics. High-scale proteomics promises to transform medicine with personalized diagnostics, prevention, and treatment. We have now reached into the human proteome to quantify more than 1000 proteins in any human matrix - serum, plasma, CSF, BAL, and also tissue extracts - with our new SOMAmer-based proteomics platform. The surprising and pleasant news is that we have made unbiased protein biomarker discovery a routine and fast exercise. The downstream implications of the platform are substantial.     

Ubiquitin, a central component of selective cytoplasmic proteolysis, is linked to proteins residing at the locus of non-selective proteolysis, the vacuole.

Ubiquitin, an evolutionary highly conserved protein, is known to be involved in selective proteolysis in the cytoplasm. Here we show that ubiquitin-protein conjugates are also found in the yeast vacuole. Mutants defective in the major vacuolar endopeptidases, proteinase yscA and yscB, lead to accumulation of ubiquitin-protein conjugates in this cellular organelle.     

Analysis of high accuracy, quantitative proteomics data in the MaxQB database

MS-based proteomics generates rapidly increasing amounts of precise and quantitative information. Analysis of individual proteomic experiments has made great strides, but the crucial ability to compare and store information across different proteome measurements still presents many challenges. For example, it has been difficult to avoid contamination of databases with low quality peptide identifications, to control for the inflation in false positive identifications when combining data sets, and to integrate quantitative data. Although, for example, the contamination with low quality identifications has been addressed by joint analysis of deposited raw data in some public repositories, we reasoned that there should be a role for a database specifically designed for high resolution and quantitative data. Here we describe a novel database termed MaxQB that stores and displays collections of large proteomics projects and allows joint analysis and comparison. We demonstrate the analysis tools of MaxQB using proteome data of 11 different human cell lines and 28 mouse tissues. The database-wide false discovery rate is controlled by adjusting the project specific cutoff scores for the combined data sets. The 11 cell line proteomes together identify proteins expressed from more than half of all human genes. For each protein of interest, expression levels estimated by label-free quantification can be visualized across the cell lines. Similarly, the expression rank order and estimated amount of each protein within each proteome are plotted. We used MaxQB to calculate the signal reproducibility of the detected peptides for the same proteins across different proteomes. Spearman rank correlation between peptide intensity and detection probability of identified proteins was greater than 0.8 for 64% of the proteome, whereas a minority of proteins have negative correlation. This information can be used to pinpoint false protein identifications, independently of peptide database scores. The information contained in MaxQB, including high resolution fragment spectra, is accessible to the community via a user-friendly web interface at http://www.biochem.mpg.de/maxqb.     

MASPECTRAS: a platform for management and analysis of proteomics LC-MS/MS data

Background  

The advancements of proteomics technologies have led to a rapid increase in the number, size and rate at which datasets are generated. Managing and extracting valuable information from such datasets requires the use of data management platforms and computational approaches.