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Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been successfully applied to elucidating biological questions trough the analysis of proteins, peptides, and nucleic acids. Here, we review the different approaches for analyzing the data that is generated by MALDI-MS. The first step in the analysis is the processing of the raw data to find peaks that correspond to the analytes. The peaks are characterized by their areas (or heights) and their centroids. The peak area can be used as a measure of the quantity of the analyte, and the centroid can be used to determine the mass of the analyte. The masses are then compared to models of the analyte, and these models are ranked according to how well they fit the data and their significance is calculated. This allows the determination of the identity (sequence and modifications) of the analytes. We show how this general data analysis workflow is applied to protein and nucleic acid chemistry as well as proteomics.

This paper describes an algorithm to apply proteotypic peptide sequence libraries to protein identifications performed using tandem mass spectrometry (MS/MS). Proteotypic peptides are those peptides in a protein sequence that are most likely to be confidently observed by current MS-based proteomics methods. Libraries of proteotypic peptide sequences were compiled from the Global Proteome Machine Database for Homo sapiens and Saccharomyces cerevisiae model species proteomes. These libraries were used to scan through collections of tandem mass spectra to discover which proteins were represented by the data sets, followed by detailed analysis of the spectra with the full protein sequences corresponding to the discovered proteotypic peptides. This algorithm (Proteotypic Peptide Profiling, or P3) resulted in sequence-to-spectrum matches comparable to those obtained by conventional protein identification algorithms using only full protein sequences, with a 20-fold reduction in the time required to perform the identification calculations. The proteotypic peptide libraries, the open source code for the implementation of the search algorithm and a website for using the software have been made freely available. Approximately 4% of the residues in the H. sapiens proteome were required in the proteotypic peptide library to successfully identify proteins.

An algorithm for reducing the time necessary to match a large set of peptide tandem mass spectra with a list of protein sequences is described. This algorithm breaks the process into multiple steps. A rapid survey step identifies all protein sequences that are reasonable candidates for a match with a set of tandem mass spectra. These candidates are then used as models, which are refined by detailed analysis of the set of tandem mass spectra for evidence of incomplete enzymatic hydrolysis, non-specific hydrolysis and chemical modifications of amino acid residues resulting from either post-translational modifications or sample handling. Compared with current one-step methods for matching proteins to mass spectra, this multiple-step method can decrease the time required for the calculation by several orders of magnitude.

RADARS, a rapid, automated, data archiving and retrieval software system for high-throughput proteomic mass spectral data processing and storage, is described. The majority of mass spectrometer data files are compatible with RADARS, for consistent processing. The system automatically takes unprocessed data files, identifies proteins via in silico database searching, then stores the processed data and search results in a relational database suitable for customized reporting. The system is robust, used in 24/7 operation, accessible to multiple users of an intranet through a web browser, may be monitored by Virtual Private Network, and is secure. RADARS is scalable for use on one or many computers, and is suited to multiple processor systems. It can incorporate any local database in FASTA format, and can search protein and DNA databases online. A key feature is a suite of visualisation tools (many available gratis), allowing facile manipulation of spectra, by hand annotation, reanalysis, and access to all procedures. We also described the use of Sonar MS/MS, a novel, rapid search engine requiring 40 MB RAM per process for searches against a genomic or EST database translated in all six reading frames. RADARS reduces the cost of analysis by its efficient algorithms: Sonar MS/MS can identifiy proteins without accurate knowledge of the parent ion mass and without protein tags. Statistical scoring methods provide close-to-expert accuracy and brings robust data analysis to the non-expert user.

Soluble A beta (Sa beta) is normally present at a low concentration in human plasma and cerebrospinal fluid. Although the factors involved in the regulation of Sa beta plasma levels are still unknown, we have explored its excretion in the urine as one of the possible homeostatic mechanisms. The presence of Sa beta in the urine was investigated via immunoprecipitation experiments with anti-A beta antibodies followed by detection and identification by immunoblot, MALDI mass spectrometry and sequence analysis. Soluble A beta (4.3 kDa) immunoreactivity was present in the urine of normal donors, Down's syndrome individuals as well as in patients with renal disorders exhibiting glomerular or mixed proteinuria. Edman degradation of the immunoprecipitated material yielded the intact A beta N-terminus and mass spectra analysis indicated the existence of a major component at mlz 4327, corresponding to the molecular mass of A beta1-40. Semiquantitative data obtained from the immunoprecipitation experiments indicate that under normal conditions the daily excretion of intact Sa beta in the urine represents less than 1% of the circulating pool.

This paper describes the application of matrix convolution filters to time-of-flight matrix-assisted laser desorption mass spectra to improve the appearance of a particular spectrum and to extract peaks from poorly resolved signals. These filters are commonly used in image enhancement to sharpen blurry images and remove noise because of their general applicability and their modest requirements for calculation speed. The theory of these filters is discussed, as applied to mass spectra, and examples of spectra processed using a variety of filters are shown to demonstrate the improvements and artifacts that can be generated.

This paper describes the fabrication and use of thin polycrystalline films in matrix-assisted laser desorption experiments. These films consist of microcrystals of protein-doped matrix (crystal dimensions ⩽ 1 μm). The films produce intense protein-ion currents and can be grown in the presence of high concentrations of involatile solvents (e.g., glycerol, 6 M urea) without any purification. They strongly adhere to the substrate, allowing easier washing of the film, compared to dried-droplet deposits. The films are also more uniform than dried-droplet deposits, with respect to ion production. It is suggested that matrix-assisted laser desorption/ionization samples are composed of nonlinear optical devices that function as polymer ion-current sources. These devices (protein-doped matrix crystals) can be designed and fabricated in many forms to serve the special functions required by the analytical scientist.

This paper describes several protocols for growing large, protein-doped 3,5-dimethoxy-4-hydroxy-trans-cinnamic acid crystals. Examination of these crystals using laser desorption shows that the mass spectra obtained from the crystals can be useful for biochemical analysis. One particular crystal growing protocol allowed a non-covalently bound heme group of horse muscle myoglobin to remain attached to the polypeptide following laser ablation and ionization. Crystals could be grown in solutions that contained involatile solvents that normally inhibit polypeptide ion production, such as glycerol. These crystals were protein doped and produced acceptable analytical mass spectra. The results suggest that some problems associated with the frequently used droplet-drying method of sample preparation are caused by the changing concentration conditions present in drying solutions.

Phenomenological models were proposed to explain the experimentally observed dependence of protein ion yields with laser fluence in matrix-assisted laser desorption. Assuming that the illuminating laser had a Gaussian intensity profile at the sample being examined, it was possible to fit the experimental points with a model that only assumes a fluence threshold for ion production. No additional dependence of protein ion yield on illuminating fluence above the threshold value was necessary to explain the data.

The paper reports the discovery of three new matrices for the matrix-assisted laser desorption of proteins. These new matrices (sinapic, ferulic and caffeic acids) are cinnamic acid derivatives that have several pratical advantages over the nicotinic acid matrices previously used. These materials form much less intense photochemically generated adduct peaks in the protein quasimolecular ion signal and the adduct peaks that are present are easier to resolve. These new matrices are produce intense protonated-molecule ions from all of the proteins (over 50) so far examined. These new matrices are also very stable in a vacuum, allowing for their convenient use in very high vacuum applications (e.g., Fourier transform ion cyclotron resonance mass spectrometry).