Response to: the problem with peptide presumption and low Mascot scoring

A letter published in January 2011, "The Problem with Peptide Presumption and Low Mascot Scoring", raised concerns about the reporting of peptide identifications based on mass spectrometry data with high precursor mass accuracy. We explain why we believe these concerns are unfounded.     

Team Spirits: The Native American Mascot Controversy

Team Spirits: The Native American Mascot Controversy. C. Richard King and Charles Fruehling Springwood. eds. Lincoln: University of Nebraska Press, 2001. 356 pp.     

Probabilistic consensus scoring improves tandem mass spectrometry peptide identification

Database search is a standard technique for identifying peptides from their tandem mass spectra. To increase the number of correctly identified peptides, we suggest a probabilistic framework that allows the combination of scores from different search engines into a joint consensus score. Central to the approach is a novel method to estimate scores for peptides not found by an individual search engine. This approach allows the estimation of p-values for each candidate peptide and their combination across all search engines. The consensus approach works better than any single search engine across all different instrument types considered in this study. Improvements vary strongly from platform to platform and from search engine to search engine. Compared to the industry standard MASCOT, our approach can identify up to 60% more peptides. The software for consensus predictions is implemented in C++ as part of OpenMS, a software framework for mass spectrometry. The source code is available in the current development version of OpenMS and can easily be used as a command line application or via a graphical pipeline designer TOPPAS.     

OLAV-PMF: a novel scoring scheme for high-throughput peptide mass fingerprinting

We propose a new type of probabilistic scoring scheme framework for protein identification from peptide masses. We first introduce the framework itself and explain its requirements. In a second part, we describe a particular implementation and test it on a data set of more than 8000 MALDI-TOF spectra with known contents. Doing so, we also compare its performance to two widely used scoring schemes, thereby demonstrating the potential of the proposed approach.     

Free trypsin-catalyzed peptide synthesis in acetonitrile with low water content

Summary The reactions of Bz-Arg-OEt, Boc-Lys-Ome and Boc-Tyr-Lys-OMe with various amino acid amides and peptides, catalyzed by free trypsin in acetonitrile in the presence of triethylamine and 5 vol. % of water, were studied. The synthesis of Bz-Arg-Leu-NH₂ was strongly dependent on the water content (from 2 to 10%) in the reaction medium. Excess of base (triethylamine) had no negative effect on the reaction. The reaction proceeded analogously also in some aliphatic alcohols but not in dimethylformamide.     

The problem with the species problem

When Charles Darwin convinced the scientific community that species evolve, the long-held essentialist view of each species as fixed was rejected and a clear conceptual understanding of the term was lost. For the next century, a real species problem existed that became culturally entrenched within the scientific community. Although largely solved decades ago, the species problem remains entrenched today due to a suite of factors. Most of the factors that help maintain its perceived intractability have been revealed and logically dismissed; yet this is not widely known so those factors continue to be influential. It is time to recognize this false foundation and relegate the species problem to history.     

The ataxia telangiectasia clastogenic factor is a low molecular weight peptide

Summary The clastogenic factor in the plasma of ataxia telangiectasia (AT) patients and in conditioned medium from AT skin fibroblast cultures is a peptide with a molecular weight in the range of 500 to 1000. No clastogenic activity could be demonstrated in extracts of cultured AT fibroblasts.     

DirecTag: accurate sequence tags from peptide MS/MS through statistical scoring

In shotgun proteomics, tandem mass spectra of peptides are typically identified through database search algorithms such as Sequest. We have developed DirecTag, an open-source algorithm to infer partial sequence tags directly from observed fragment ions. This algorithm is unique in its implementation of three separate scoring systems to evaluate each tag on the basis of peak intensity, m/ z fidelity, and complementarity. In data sets from several types of mass spectrometers, DirecTag reproducibly exceeded the accuracy and speed of InsPecT and GutenTag, two previously published algorithms for this purpose. The source code and binaries for DirecTag are available from http://fenchurch.mc.vanderbilt.edu.     

Predicting peptide binding affinities to MHC molecules using a modified semi-empirical scoring function

The Major Histocompatibility Complex (MHC) plays an important role in the human immune system. The MHC is involved in the antigen presentation system assisting T cells to identify foreign or pathogenic proteins. However, an MHC molecule binding a self-peptide may incorrectly trigger an immune response and cause an autoimmune disease, such as multiple sclerosis. Understanding the molecular mechanism of this process will greatly assist in determining the aetiology of various diseases and in the design of effective drugs. In the present study, we have used the Fresno semi-empirical scoring function and modify the approach to the prediction of peptide-MHC binding by using open-source and public domain software. We apply the method to HLA class II alleles DR15, DR1, and DR4, and the HLA class I allele HLA A2. Our analysis shows that using a large set of binding data and multiple crystal structures improves the predictive capability of the method. The performance of the method is also shown to be correlated to the structural similarity of the crystal structures used. We have exposed some of the obstacles faced by structure-based prediction methods and proposed possible solutions to those obstacles. It is envisaged that these obstacles need to be addressed before the performance of structure-based methods can be on par with the sequence-based methods.     

Docking and scoring with alternative side-chain conformations

We describe a scoring and modeling procedure for docking ligands into protein models that have either modeled or flexible side-chain conformations. Our methodical contribution comprises a procedure for generating new potentials of mean force for the ROTA scoring function which we have introduced previously for optimizing side-chain conformations with the tool IRECS. The ROTA potentials are specially trained to tolerate small-scale positional errors of atoms that are characteristic of (i) side-chain conformations that are modeled using a sparse rotamer library and (ii) ligand conformations that are generated using a docking program. We generated both rigid and flexible protein models with our side-chain prediction tool IRECS and docked ligands to proteins using the scoring function ROTA and the docking programs FlexX (for rigid side chains) and FlexE (for flexible side chains). We validated our approach on the forty screening targets of the DUD database. The validation shows that the ROTA potentials are especially well suited for estimating the binding affinity of ligands to proteins. The results also show that our procedure can compensate for the performance decrease in screening that occurs when using protein models with side chains modeled with a rotamer library instead of using X-ray structures. The average runtime per ligand of our method is 168 seconds on an Opteron V20z, which is fast enough to allow virtual screening of compound libraries for drug candidates.     

SCOPE: a probabilistic model for scoring tandem mass spectra against a peptide database

Proteomics, or the direct analysis of the expressed protein components of a cell, is critical to our understanding of cellular biological processes in normal and diseased tissue. A key requirement for its success is the ability to identify proteins in complex mixtures. Recent technological advances in tandem mass spectrometry has made it the method of choice for high-throughput identification of proteins. Unfortunately, the software for unambiguously identifying peptide sequences has not kept pace with the recent hardware improvements in mass spectrometry instruments. Critical for reliable high-throughput protein identification, scoring functions evaluate the quality of a match between experimental spectra and a database peptide. Current scoring function technology relies heavily on ad-hoc parameterization and manual curation by experienced mass spectrometrists. In this work, we propose a two-stage stochastic model for the observed MS/MS spectrum, given a peptide. Our model explicitly incorporates fragment ion probabilities, noisy spectra, and instrument measurement error. We describe how to compute this probability based score efficiently, using a dynamic programming technique. A prototype implementation demonstrates the effectiveness of the model.     

Modification of antimicrobial peptide with low molar mass poly(ethylene glycol)

PEGylation of peptide drugs prolongs their circulating lifetimes in plasma. However, PEGylation can produce a decrease in the in vitro bioactivity. Longer poly(ethylene glycol) (PEG) chains are favourable for circulating lifetimes but unfavourable for in vitro bioactivities. In order to circumvent the conflicting effects of PEG length, a hydrophobic peptide, using an antimicrobial peptide as a model, was PEGylated with short PEG chains. The PEGylated peptides self-assembled in aqueous solution into micelles with PEG shell and peptide core. In these micelles, the core peptides were protected by the shell, thus reducing proteolytic degradation. Meanwhile, most of the in vitro antimicrobial activities still remained due to the short PEG chain attached. The stabilities of the PEGylated peptides were much higher than that of the unPEGylated peptides in the presence of chymotrypsin and serum. The antimicrobial activities of the PEGylated peptides in the presence of serum, an ex vivo assay, were much higher than that of the unPEGylated peptide.