Retention time alignment algorithms for LC/MS data must consider non-linear shifts.

MOTIVATION: Proteomics has particularly evolved to become of high interest for the field of biomarker discovery and drug development. Especially the combination of liquid chromatography and mass spectrometry (LC/MS) has proven to be a powerful technique for analyzing protein mixtures. Clinically orientated proteomic studies will have to compare hundreds of LC/MS runs at a time. In order to compare different runs, sophisticated preprocessing steps have to be performed. An important step is the retention time (rt) alignment of LC/MS runs. Especially non-linear shifts in the rt between pairs of LC/MS runs make this a crucial and non-trivial problem. RESULTS: For the purpose of demonstrating the particular importance of correcting non-linear rt shifts, we evaluate and compare different alignment algorithms. We present and analyze two versions of a new algorithm that is based on regression techniques, once assuming and estimating only linear shifts and once also allowing for the estimation of non-linear shifts. As an example for another type of alignment method we use an established alignment algorithm based on shifting vectors that we adapted to allow for correcting non-linear shifts also. In a simulation study, we show that rt alignment procedures that can estimate non-linear shifts yield clearly better alignments. This is even true under mild non-linear deviations. AVAILABILITY: R code for the regression-based alignment methods and simulated datasets are available at http://www.statistik.tu-dortmund.de/genetik-publikationen-alignment.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Study of posttranslational modifications in lenticular alphaA-Crystallin of mice using proteomic analysis techniques.

In the present work the complexity in the 2D-gel protein pattern of murin lenticular alphaA-Crystallin was analyzed. An in depth study of the different protein isoforms was done combining different proteomic tools. Lens proteins of four different ages, from embryo to 100-week-old mice, were separated by large 2D-PAGE, revealing an increase in the number and intensity of the spots of alphaA-Crystallin during the process of aging. For further analyses the oldest mice were chosen. Comparison and evaluation of two different staining methods proved Imidazole-Zinc to be a good alternative to the generally used Coomassie stain. The characterization of the different alphaA-Crystallin protein species was done using nanoLC-ESI-MS/MS (liquid chromatography electrospray ionisation tandem mass spectrometry). Data interpretation was done by database searching, manual validation and a new MS/MS-interpretation tool for posttranslational modifications--the PTM-Explorer. Using this way, eight different phosphorylation sites were identified and localized; the identification of four of them was not published so far. Furthermore, quantitative N-terminal acetylation of alphaA-Crystallin and variable C-terminal truncation was observed, also not published in this extent yet. The results of the mass spectrometric analysis were validated by immunoblotting experiments using two different alphaA-Crystallin specific antibodies. In addition, a fluorescent phospho-specific stain was used to detect the protein spots including phosphorylation groups. Re-separation 2D-PAGE was done to round off the present study and explain the appearance of some of the protein spots in the gel as artifacts of the 2D-PAGE separation.

Gaining knowledge from previously unexplained spectra-application of the PTM-Explorer software to detect PTM in HUPO BPP MS/MS data.

A novel software tool named PTM-Explorer has been applied to LC-MS/MS datasets acquired within the Human Proteome Organisation (HUPO) Brain Proteome Project (BPP). PTM-Explorer enables automatic identification of peptide MS/MS spectra that were not explained in typical sequence database searches. The main focus was detection of PTMs, but PTM-Explorer detects also unspecific peptide cleavage, mass measurement errors, experimental modifications, amino acid substitutions, transpeptidation products and unknown mass shifts. To avoid a combinatorial problem the search is restricted to a set of selected protein sequences, which stem from previous protein identifications using a common sequence database search. Prior to application to the HUPO BPP data, PTM-Explorer was evaluated on excellently manually characterized and evaluated LC-MS/MS data sets from Alpha-A-Crystallin gel spots obtained from mouse eye lens. Besides various PTMs including phosphorylation, a wealth of experimental modifications and unspecific cleavage products were successfully detected, completing the primary structure information of the measured proteins. Our results indicate that a large amount of MS/MS spectra that currently remain unidentified in standard database searches contain valuable information that can only be elucidated using suitable software tools.

The power of cooperative investigation: summary and comparison of the HUPO Brain Proteome Project pilot study results.

Within the pilot phase of the HUPO Brain Proteome Project, nine participating laboratories analysed human (epilepsy and/or post mortem material) and mouse brain samples (embryonic, juvenile and adult), respectively, using a variety of different state of the art techniques. Thirty-seven different analytical approaches were accomplished. Of these analyses, 17 were done differentially, i.e. the protein expression patterns of the different samples (human or mouse) were compared. A catalogue of all proteins present in the respective sample was built in 20 analyses (mapping). All data were collected in the Data Collection Center in Bochum, Germany, and were reprocessed according to thoroughly defined parameters. In this report, a summary of all results and inter-laboratory comparisons with respect to the number of identified proteins, the analysed organism, and the used techniques is presented.

Automated reprocessing pipeline for searching heterogeneous mass spectrometric data of the HUPO Brain Proteome Project pilot phase.

The newly available techniques for sensitive proteome analysis and the resulting amount of data require a new bioinformatics focus on automatic methods for spectrum reprocessing and peptide/protein validation. Manual validation of results in such studies is not feasible and objective enough for quality relevant interpretation. The necessity for tools enabling an automatic quality control is, therefore, important to produce reliable and comparable data in such big consortia as the Human Proteome Organization Brain Proteome Project. Standards and well-defined processing pipelines are important for these consortia. We show a way for choosing the right database model, through collecting data, processing these with a decoy database and end up with a quality controlled protein list merged from several search engines, including a known false-positive rate.

Tryptic transpeptidation products observed in proteome analysis by liquid chromatography-tandem mass spectrometry.

Commonly, prior to mass spectrometry based analysis of proteins or protein mixtures, the proteins are subjected to specific enzymatic proteolysis. For this purpose trypsin is most frequently used. However, the process of proteolysis is not unflawed. For example, some side activities of trypsin are known and have already been described in the literature (e.g., chymotryptic activity). Here, we describe the occurrence of transpeptidated peptides during standard proteome analysis using two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometric protein identification. Different types of transpeptidated peptides have been detected. The most frequently observed transpeptidation reaction is N-terminal addition of arginine or lysine to peptides. Furthermore, addition of two amino acids to the N-terminus of a peptide has also been detected. Another transpeptidation that we observed, is combination of two peptides, which were originally located in different regions of the analyzed protein. Currently, the full amount of peptides generated by transpeptidation is not clear. However, it should be recognized that protein information is presently lost as these effects are not detectable with available database search software.

Evaluation of algorithms for protein identification from sequence databases using mass spectrometry data.

In this work, the commonly used algorithms for mass spectrometry based protein identification, Mascot, MS-Fit, ProFound and SEQUEST, were studied in respect to the selectivity and sensitivity of their searches. The influence of various search parameters were also investigated. Approximately 6600 searches were performed using different search engines with several search parameters to establish a statistical basis. The applied mass spectrometric data set was chosen from a current proteome study. The huge amount of data could only be handled with computational assistance. We present a software solution for fully automated triggering of several peptide mass fingerprinting (PMF) and peptide fragmentation fingerprinting (PFF) algorithms. The development of this high-throughput method made an intensive evaluation based on data acquired in a typical proteome project possible. Previous evaluations of PMF and PFF algorithms were mainly based on simulations.

Interpretation of mass spectrometry data for high-throughput proteomics.

Recent developments in proteomics have revealed a bottleneck in bioinformatics: high-quality interpretation of acquired MS data. The ability to generate thousands of MS spectra per day, and the demand for this, makes manual methods inadequate for analysis and underlines the need to transfer the advanced capabilities of an expert human user into sophisticated MS interpretation algorithms. The identification rate in current high-throughput proteomics studies is not only a matter of instrumentation. We present software for high-throughput PMF identification, which enables robust and confident protein identification at higher rates. This has been achieved by automated calibration, peak rejection, and use of a meta search approach which employs various PMF search engines. The automatic calibration consists of a dynamic, spectral information-dependent algorithm, which combines various known calibration methods and iteratively establishes an optimised calibration. The peak rejection algorithm filters signals that are unrelated to the analysed protein by use of automatically generated and dataset-dependent exclusion lists. In the "meta search" several known PMF search engines are triggered and their results are merged by use of a meta score. The significance of the meta score was assessed by simulation of PMF identification with 10,000 artificial spectra resembling a data situation close to the measured dataset. By means of this simulation the meta score is linked to expectation values as a statistical measure. The presented software is part of the proteome database ProteinScape which links the information derived from MS data to other relevant proteomics data. We demonstrate the performance of the presented system with MS data from 1891 PMF spectra. As a result of automatic calibration and peak rejection the identification rate increased from 6% to 44%.

Alterations in the mouse and human proteome caused by Huntington's disease.

Huntington's disease is an autosomal dominantly inherited disease that usually starts in midlife and inevitably leads to death. In our effort to identify proteins involved in processes upstream or downstream of the disease-causing huntingtin, we studied the proteome of a well established mouse model by large gel two-dimensional electrophoresis. We could demonstrate for the first time at the protein level that alpha1-antitrypsin and alphaB-crystalline both decrease in expression over the course of disease. Importantly, the alpha1-antitrypsin decrease in the brain precedes that in liver and testes in mice. Reduced expression of the serine protease inhibitors alpha1-antitrypsin and contraspin was found in liver, heart, and testes close to terminal disease. Decreased expression of the chaperone alphaB-crystallin was found exclusively in the brain. In three brain regions obtained post-mortem from Huntington's disease patients, alpha1-antitrypsin expression was also altered. Reduced expression of the major urinary proteins not found in the brain was seen in the liver of affected mice, demonstrating that the disease exerts its influence outside the brain of transgenic mice at the protein level. Maintaining alpha1-antitrypsin and alphaB-crystallin availability during the course of Huntington's disease might prevent neuronal cell death and therefore could be useful in delaying the disease progression.