Laser desorption ionization-time-of-flight mass analysis of perfluoropolyether monolayer directly from hard disk medium surface.

Modern life is dependent on computer technology, and because the volume of digital data in the world is increasing rapidly, the importance of data storage devices is also increasing rapidly. Among them, demands for magnetic disk drive well-known as hard disk drives is quite huge and information recording density on the disk media is continuing to grow dramatically. For the research and development of the magnetic disk media, it is critical to investigate and characterize the lubricant layer formed on the disk media surface. However, it is difficult because the layer is only a monolayer which has only approximately 1 nm thickness in many cases. Although matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) measurements of monolayers have already been reported (Su, J.; Mrksich, M. Langmuir, 2003, 19, 4867-4870), lubricants used here are (co)polymers which have molecular weight distributions and are mixtures of various degrees of polymerization. This can reduce the sensitivity of MS measurement because the number (or density) of distinct single molecular species is lower than for homogeneous samples. In this report, direct measurement and characterization of lubricant monolayers using the LDI-TOF-MS instrument is performed to gain insight into detailed information like average molecular weight, polymer distribution, and two-dimensional mapping directly from magnetic disk monolayers. To our knowledge, this is the first time such information was acquired directly from hard disk media. The technique reported here might open up new possibilities also for investigations of various electronic devices other than magnetic hard disks.

Protein Information and Knowledge Extractor: Discovering biological information from proteomics data.

One of the main goals in proteomics is to solve biological and molecular questions regarding a set of identified proteins. In order to achieve this goal, one has to extract and collect the existing biological data from public repositories for every protein and afterward, analyze and organize the collected data. Due to the complexity of this task and the huge amount of data available, it is not possible to gather this information by hand, making it necessary to find automatic methods of data collection. Within a proteomic context, we have developed Protein Information and Knowledge Extractor (PIKE) which solves this problem by automatically accessing several public information systems and databases across the Internet. PIKE bioinformatics tool starts with a set of identified proteins, listed as the most common protein databases accession codes, and retrieves all relevant and updated information from the most relevant databases. Once the search is complete, PIKE summarizes the information for every single protein using several file formats that share and exchange the information with other software tools. It is our opinion that PIKE represents a great step forward for information procurement and drastically reduces manual database validation for large proteomic studies. It is available at http://proteo.cnb.csic.es/pike.

Mass spectrometric top-down analysis of proteins.

The comprehensive analysis of intact proteins down to the level of their individual amino acid sequence and the entirety of post-translational modifications is an area that can hardly be covered by the typical workflow in MS based protein analysis, which comprises enzymatic digestion, mass spectrometric analysis and subsequent database search. This approach typically provides 20-80% sequence coverage, which is not sufficient for the characterization of biopharmaceuticals, for example. This generates the requirement for a comprehensive analysis of the protein, without the risk of losing sequence information due to undetected peptides. Top-down sequencing of proteins starts from the intact protein, typically by determining the intact protein mass in the first step, a fragmentation of the intact protein is then performed within the mass spectrometer, resulting in fragment ions that allow us to pinpoint the protein sequence, as well as potential modifications or mutations in their localization and structure. A number of technologies have been developed for this task in the last few years, based on various different mass spectrometric instrument configurations, but typically based on the same technology platforms as used for bottom-up strategies. Thus, the use of one specific instrument often allows the application of top-down and bottom-up technologies in a complementary way, providing much more detailed information about the proteins of interest than either of the approaches alone.

Initiation of mammalian O-mannosylation in vivo is independent of a consensus sequence and controlled by peptide regions within and upstream of the alpha-dystroglycan mucin domain.

To reveal insight into the initiation of mammalian O-mannosylation in vivo, recombinant glycosylation probes containing sections of human alpha-dystroglycan (hDG) were expressed in epithelial cell lines. We demonstrate that O-mannosylation within the mucin domain of hDG occurs preferentially at Thr/Ser residues that are flanked by basic amino acids. Protein O-mannosylation is independent of a consensus sequence, but strictly dependent on a peptide region located upstream of the mucin domain. This peptide region cannot be replaced by other N-terminal peptides, however, it is not sufficient to induce O-mannosylation on a structurally distinct mucin domain in hybrid constructs. The presented in vivo evidence for a more complex regulation of mammalian O-mannosylation contrasts with a recent in vitro study of O-mannosylation in human alpha-dystroglycan peptides indicating the existence of an 18-meric consensus sequence. We demonstrate in vivo that the entire region p377-417 is necessary and sufficient for O-mannosylation initiation of hDG, but not of MUC1 tandem repeats. The feature of a doubly controlled initiation process distinguishes mammalian O-mannosylation from other types of O-glycosylation, which are largely controlled by structural properties of the substrate positions and their local peptide environment.

The minimum information about a proteomics experiment (MIAPE).

Both the generation and the analysis of proteomics data are now widespread, and high-throughput approaches are commonplace. Protocols continue to increase in complexity as methods and technologies evolve and diversify. To encourage the standardized collection, integration, storage and dissemination of proteomics data, the Human Proteome Organization's Proteomics Standards Initiative develops guidance modules for reporting the use of techniques such as gel electrophoresis and mass spectrometry. This paper describes the processes and principles underpinning the development of these modules; discusses the ramifications for various interest groups such as experimentalists, funders, publishers and the private sector; addresses the issue of overlap with other reporting guidelines; and highlights the criticality of appropriate tools and resources in enabling 'MIAPE-compliant' reporting.

The Geodia cydonium galectin exhibits prototype and chimera-type characteristics and a unique sequence polymorphism within its carbohydrate recognition domain.

The ancestral galectin from the sponge Geodia cydonium (GCG) is classified on a structural basis to the prototype subfamily, whereas its carbohydrate-binding specificity is related to that of the mammalian chimera-type galectin-3. This dual coordination reveals GCG as a potential precursor of the later evolved galectin subfamilies, which is reflected in the primary structure of the protein. This study provides evidence that GCG is the LECT1 gene product, while neither a previously described LECT2 gene nor a functional LECT2 gene product was found in the specimen under investigation. The electrophoretically separated protein isomers with apparent molecular masses of 13, 15, and 16 kDa correspond to variants of the LECT1 protein-exhibiting peptide sequence polymorphisms that concern critical positions of the carbohydrate recognition domain (13 kDa: Leu51, Asn55, His130, Gly137; 15 kDa: Ser51, Asn55, Asn130, Gly137; 16 kDa: Ser51, Tyr55, Asn130, Glu137). Four residues, highly conserved in the galectin family, are substituted. None of the residues claimed to be involved in interactions with GalNAcalpha1-3 moieties at an extended binding subsite of galectin-3 was identified in the corresponding positions of GCG. Apparently, the substitutions do not confer distinct binding characteristics to the GCG variants as evidenced by binding studies with a recombinantly expressed 15-kDa isoform. The natural isoforms as well as the recombinant 15-kDa isoform oligomerize by the formation of non-covalent heteromeric or homomeric complexes. A phosphorylation of the galectin was confirmed neither by mass spectrometry nor by alkaline phosphatase treatment combined with isoelectric focusing.

Comparison of laser-induced dissociation and high-energy collision-induced dissociation using matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) for peptide and protein identification.

The fragmentation of peptides under laser-induced dissociation (LID) as well as high-energy collision-induced dissociation (CID) conditions has been investigated. The effect of the different fragmentation mechanisms on the formation of specific fragment ion types and the usability of the resulting spectra, e.g. for high-throughput protein identification, has been evaluated. Also, basic investigations on the influence of the matrix, as well as laser fluence, on the fragment ion formation and the consequences in the spectral appearance are discussed. The preconditions for obtaining 'pure' CID spectra on matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) instruments are evaluated and discussed as well as the differences between LID and CID in the resulting fragment ion types. While containing a wealth of information due to additional fragment ions in comparison with LID, CID spectra are significantly more complex than LID spectra and, due to different fragmentation patterns, the CID spectra are of limited use for protein identification, even under optimized parameter settings, due to significantly lower scores for the individual spectra. Conditions for optimal results regarding protein identification using MALDI-TOF/TOF instruments have been evaluated. For database searches using tandem mass spectrometric data, the use of LID as fragmentation technique in combination with parameter settings supporting the use of internal fragment ions turned out to yield the optimal results.

"Affinity-proteomics": direct protein identification from biological material using mass spectrometric epitope mapping.

We describe here a new approach for the identification of affinity-bound proteins by proteolytic generation and mass spectrometric analysis of their antibody bound epitope peptides (epitope excision). The cardiac muscle protein troponin T was chosen as a protein antigen because of its diagnostic importance in myocardial infarct, and its previously characterised epitope structure. Two monoclonal antibodies (IgG1-1B10 and IgG1-11.7) raised against intact human troponin T were found to be completely cross reactive with bovine heart troponin T. A combination of immuno-affinity isolation, partial proteolytic degradation (epitope excision), mass spectrometric peptide mapping, and database analysis was used for the direct identification of Tn T from bovine heart cell lysate. Selective binding of the protein was achieved by addition of bovine heart cell lysate to the Sepharose-immobilised monoclonal antibodies, followed by removal of supernatant material containing unbound protein. While still bound to the affinity matrix the protein was partially degraded thereby generating a set of affinity-bound, overlapping peptide fragments comprising the epitope. Following dissociation from the antibody the epitope peptides were analysed by matrix assisted laser desorption-ionisation (MALDI) and electrospray-ionisation (ESI) mass spectrometry. The peptide masses identified by mass spectrometry were used to perform an automated database search, combined with a search for a common "epitope motif". This procedure resulted in the unequivocal identification of the protein from biological material with only a minimum number of peptide masses, and requiring only limited mass-determination accuracy. The dramatic increase of selectivity for identification of the protein by combining the antigen-antibody specificity with the redundancy of peptide sequences renders this "affinity-proteomics" approach a powerful tool for mass spectrometric identification of proteins from biological material.

A robust approach for the analysis of peptides in the low femtomole range by capillary electrophoresis-tandem mass spectrometry.

A capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) approach has been developed for routine application in proteomic studies. Robustness of the coupling is achieved by using a standard coaxial sheath-flow sprayer. Thereby, greater stability than nanoelectrospray ionization-mass spectrometry coupling of sheathless capillary electrophoresis or nanoliquid chromatography (nano-LC) is achieved, resulting in stable operation for several weeks and unattended overnight sequences. The applied sheath flow is reduced to 1-2 microL/min in order to increase sensitivity. Standard peptides and those of digests of standard proteins and gel-separated proteins can be detected in the low femtomole range (full scan and MS/MS). Detection limits are found to be as low as 500 amol. Low femtomole amounts are required for unequivocal identification by MS/MS experiments in the ion trap and subsequent database search. By applying a simple pH-mediated stacking the concentration sensitivity can be lowered to some tens of fmol/microL (nM), depending on capillary size. This sensitivity is close to published values for sheathless CE-MS and nano-LC-MS, respectively (a comparison to reference values is presented). Moreover, with capillaries of about 50 cm in length separations in less than 10 min are possible resulting in a throughput of up to four analyses per hour. This is a factor of 4-12 times faster than nano-LC separation, being the state-of-the-art techniques for proteomic studies.

Identification and discrimination of Staphylococcus aureus strains using matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

Staphylococcus aureus is an important human pathogen frequently resistant to a wide range of antibiotics. Methicillin-resistant S. aureus (MRSA) strains are common nosocomial pathogens that pose a world-wide problem. Rapid and accurate discrimination between methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus is essential for appropriate therapeutic management and timely intervention for infection control. We report here the application of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) for monitoring the bacterial fingerprints expressed by two well characterized S. aureus strains ATCC 29213 (MSSA) and ATCC 43330 (MRSA). Consistent strain-specific data were obtained from subcultures analyzed over a period of three months as well as after changing the growth media from Mueller-Hinton to blood agar indicating the reliability of the method. The bacterial fingerprints of these two strains were compared to independent clinical isolates of S. aureus. A uniform signature profile for MRSA could not be identified. However, the bacterial fingerprints obtained proved to be specific for any given strain. This study demonstrates that MALDI-TOF MS is a powerful method for rapid identification of clonal strains of S. aureus, which might be useful for tracking nosocomial outbreaks of MRSA and for epidemiologic studies of infections diseases in general.