The application of mass spectrometry imaging (MS imaging) is rapidly growing with a constantly increasing number of different instrumental systems and software tools. The data format imzML was developed to allow the flexible and efficient exchange of MS imaging data between different instruments and data analysis software. imzML data is divided in two files which are linked by a universally unique identifier (UUID). Experimental details are stored in an XML file which is based on the HUPO-PSI format mzML. Information is provided in the form of a 'controlled vocabulary' (CV) in order to unequivocally describe the parameters and to avoid redundancy in nomenclature. Mass spectral data are stored in a binary file in order to allow efficient storage. imzML is supported by a growing number of software tools. Users will be no longer limited to proprietary software, but are able to use the processing software best suited for a specific question or application. MS imaging data from different instruments can be converted to imzML and displayed with identical parameters in one software package for easier comparison. All technical details necessary to implement imzML and additional background information is available at www.imzml.org.

A new scanning microprobe matrix-assisted laser desorption/ionization (SMALDI) ion source for high spatial resolution has been developed for linear ion trap and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). The source is fully compatible with commercial ion trap flanges (such as the LTQ series, Thermo Fisher Scientific). The source is designed for atmospheric pressure (AP) operation but is also suitable for mid-pressure operation. The AP mode is especially useful for investigating volatile compounds. The source can be interchanged with other ion sources within a minute when operated in the AP mode. Combining high-lateral resolution MALDI imaging with high mass resolution and high mass accuracy mass spectrometry, available in the FT-ICR mode, provides a new quality of analytical information, e.g. from biological samples. First results obtained with the new ion source demonstrate a maximum lateral resolution of 0.6 by 0.5 microm. Depending on the limit of detection of the chosen mass analyzer, however, the size of the focus had to be enlarged to a diameter of up to 8 microm in the FT-ICR mode, in order to create enough ions for detection. Mass spectra acquired for analytical imaging were obtained from single laser pulses per pixel in all the experiments. This mode allows us to investigate biological thin sections with desorption focus diameters in the micrometer range, known to cause complete evaporation of material under the laser focus with a very limited number of laser pulses. As a first example, peptide samples deposited in microstructures were investigated with the new setup. A high quality and validity of the acquired images were obtained in the ion trap mode due to the low limit of detection. High mass resolution and accuracy but poorer image quality were obtained in the ICR mode due to the lower detection sensitivity of the ICR detector.

Mass spectrometric imaging (MSI) techniques are of growing interest for the Life Sciences. In recent years, the development of new instruments employing ion sources that are tailored for spatial scanning allowed the acquisition of large data sets. A subsequent data processing, however, is still a bottleneck in the analytical process, as a manual data interpretation is impossible within a reasonable time frame. The transformation of mass spectrometric data into spatial distribution images of detected compounds turned out to be the most appropriate method to visualize the results of such scans, as humans are able to interpret images faster and easier than plain numbers. Image generation, thus, is a time-consuming and complex yet very efficient task. The free software package "Mirion," presented in this paper, allows the handling and analysis of data sets acquired by mass spectrometry imaging. Mirion can be used for image processing of MSI data obtained from many different sources, as it uses the HUPO-PSI-based standard data format imzML, which is implemented in the proprietary software of most of the mass spectrometer companies. Different graphical representations of the recorded data are available. Furthermore, automatic calculation and overlay of mass spectrometric images promotes direct comparison of different analytes for data evaluation. The program also includes tools for image processing and image analysis.

Imaging mass spectrometry is the method of scanning a sample of interest and generating an "image" of the intensity distribution of a specific analyte. The data sets consist of a large number of mass spectra which are usually acquired with identical settings. Existing data formats are not sufficient to describe an MS imaging experiment completely. The data format imzML was developed to allow the flexible and efficient exchange of MS imaging data between different instruments and data analysis software.For this purpose, the MS imaging data is divided in two separate files. The mass spectral data is stored in a binary file to ensure efficient storage. All metadata (e.g., instrumental parameters, sample details) are stored in an XML file which is based on the standard data format mzML developed by HUPO-PSI. The original mzML controlled vocabulary was extended to include specific parameters of imaging mass spectrometry (such as x/y position and spatial resolution). The two files (XML and binary) are connected by offset values in the XML file and are unambiguously linked by a universally unique identifier. The resulting datasets are comparable in size to the raw data and the separate metadata file allows flexible handling of large datasets.Several imaging MS software tools already support imzML. This allows choosing from a (growing) number of processing tools. One is no longer limited to proprietary software, but is able to use the processing software which is best suited for a specific question or application. On the other hand, measurements from different instruments can be compared within one software application using identical settings for data processing. All necessary information for evaluating and implementing imzML can be found at http://www.imzML.org .

Accurate mass values as obtainable by Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) were employed in a theoretical study to differentiate between nonmodified and phosphorylated peptides. It was found that for peptide masses up to 1000 u more than 98% of all theoretical monophosphorylated peptides (all possible combinations of proteinogenic amino acids having one phosphorylation on S, T, or Y) can be distinguished from nonphosphorylated peptides directly by their mass, if mass values are determined with an accuracy of better than Ϯ0.1 ppm. At a peptide mass of 1500 u still 70% of all possible monophosphorylated peptides are distinguishable from nonmodified peptides by their accurate mass alone. In contrast to established techniques of data-dependent multidimensional mass spectrometry, only the mass of the precursor ion is necessary to decide upon subsequent fragment ion analysis of a peptide for sequence analysis in an LC-MS/MS investigation of a complex sample, when using a precalculated mass distribution A ccurate masses of molecules or molecular ions build up a discrete structure in mass space, as a result of the mass defect of the isotopes they are composed of. Masses of atoms are therefore not the sum of masses of a certain number of nuclear protons and neutrons plus the sum of masses of electrons, but include an additional mass deficit due to the specific nuclear binding energies of the atom. As a result, each isotopic species carries a characteristic mass signature expressed as an isotope-specific accurate mass value and furthermore, each molecule carries digital signatures (i.e., number and accurate mass) of their atomic building blocks. Regarding the mass space of different substance classes such as peptides, carbohydrates, or lipids, varying ratios of their common elements (CHNOS) lead to accurate-mass distributions, which do not necessarily overlap and therefore can eventually be used for substance class identification (Figure 1a). If elemental compositions of different substance class molecules are identical accidentally, however, accurate masses are identical and cannot be resolved even with the highest mass resolving power.When introducing a new element, on the other hand, accurate mass distributions definitely become distinguishable, since the mass defect of the added element results in a new signature of the accurate masses of the molecules. This is the case, for example, when comparing nonmodified and phosphorylated peptides. With a sufficiently high mass resolving power, such species can always be distinguished from each other. As instruments for determining molecular masses are getting more and more accurate with higher and higher mass resolution, investigation of this structure is becoming an important and fundamental scientific goal. Today, mass accuracies in the range of Ϯ0.2 ppm can routinely be achieved with internal calibration on ion trap Fourier transform ion cyclotron resonance mass spectrometers [1][2][3][4], and it is quite likely that the Ϯ0.1 ppm range of m...