A new denoising and peak picking algorithm (MEND, matched filtration with experimental noise determination) for analysis of LC-MS data is described. The algorithm minimizes both random and chemical noise in order to determine MS peaks corresponding to sample components. Noise characteristics in the data set are experimentally determined and used for efficient denoising. MEND is shown to enable low-intensity peaks to be detected, thus providing additional useful information for sample analysis. The process of denoising, performed in the chromatographic time domain, does not distort peak shapes in the m/z domain, allowing accurate determination of MS peak centroids, including low-intensity peaks. MEND has been applied to denoising of LC-MALDI-TOF-MS and LC-ESI-TOF-MS data for tryptic digests of protein mixtures. MEND is shown to suppress chemical and random noise and baseline fluctuations, as well as filter out false peaks originating from the matrix (MALDI) or mobile phase (ESI). In addition, MEND is shown to be effective for protein expression analysis by allowing selection of a large number of differentially expressed ICAT pairs, due to increased signal-to-noise ratio and mass accuracy.

We previously introduced a vacuum deposition interface for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF MS) on a moving surface (e.g., quartz wheel, Mylar tape, metal target). In our present work, the approach has been extended to demonstrate parallel analysis for multiple on-line infusion MALDI MS and capillary array electrophoresis (CAE)-MALDI MS. In the infusion mode, individual peptide samples were simultaneously deposited on a Mylar tape cartridge using an array of eight capillaries, yielding eight parallel traces. For CAE-MALDI/TOF MS, the same number of separation capillaries were coupled with an array of eight infusion capillaries using a common liquid junction, containing matrix solution. A fast-scanning mirror was employed to traverse the beam of the desorption laser across the Mylar tape to probe one trace at a time. The positions of the eight sample traces formed on the tape were automatically determined, and all samples were analyzed in rapid sequence using a kilohertz repetition rate laser and a high-throughput data acquisition system. The instrumentation was operated with CAE MS for high-throughput analysis without compromising data quality. The principles of parallel separation-vacuum deposition should be generally applicable to MALDI/TOF MS analysis for proteomics and other areas where separation and high throughput are required.

The goal of this study was the development of N-terminal tags to improve peptide identification using high-throughput MALDI-TOF MS and MS/MS. The proposed tags, commercially available fluorescent derivatives of coumarin, can be advantageous for peptide analysis in both MS and MS/MS modes. This paper, part 1, will focus on the influence of derivatization on the intensities of MALDI-TOF MS signals of peptides. Labeling peptides with tags containing the coumarin core was found to enhance the intensities of peptide peaks (in some cases over 40-fold) in MALDI-TOF MS using CHCA and 2,5-DHAP matrixes. The signal enhancement was found to be peptide- and matrix-dependent, being the most pronounced for hydrophilic peptides. No correlation was found between the UV absorptivity of the tags at the excitation wavelengths typical for UV-MALDI and the magnitude of the signal enhancement. Interestingly, peptides labeled with Alexa Fluor 350, a coumarin derivative containing a sulfo group (i.e., bearing strong negative charge), showed a 5-15-fold increase in intensity of MALDI MS signal in the positive ion mode, relative to the underivatized peptides, when 2,5-DHAP was used as the matrix. The Alexa Fluor 350 tag yielded a significantly higher signal relative to that for the CAF tag, likely due to the increased hydrophobicity of the coumarin structure. With 2,5-DHB, a decrease of MALDI MS signal was observed for all coumarin-labeled peptides, again relative to the unlabeled species. These findings support the hypothesis that derivatization with coumarin, a relatively hydrophobic structure, improves incorporation of hydrophilic peptides into hydrophobic MALDI matrixes, such as CHCA and 2,5-DHAP.

High-speed, high-resolution LC separations, using a poly(styrene-divinylbenzene) monolithic column, have been coupled to MALDI MS and MS/MS through an off-line continuous deposition interface. The LC eluent was mixed with alpha-cyano-4-hydroxycinnamic acid matrix solution and deposited on a MALDI plate that had been precoated with nitrocellulose. Deposition at subatmospheric pressure (80 Torr) formed a 250-microm-wide serpentine trace with uniform width and microcrystalline morphology. The deposited trace was then analyzed in the MS mode using a MALDI-TOF/TOF MS instrument. Continuous deposition allowed interrogation of the separation with a high data sampling rate in the chromatographic dimensions, thus preserving the high resolution of narrow peaks (3-5-s peak width at half-height) of the fast monolithic LC. No extracolumn band broadening due to the deposition process was observed. Over 2000 components were resolved in a 10-min linear gradient separation of the model sample, and 386 unique peptides were identified in the subsequent MS/MS analysis. The continuous deposition interface allows the coupling of high-resolution separations to MALDI MS without degradation in separation efficiency, thus enabling high-throughput proteome analysis.

Matrix-assisted laser/desorption ionization (MALDI) mass spectrometry imaging (MSI) is widely used as a unique tool to record the distribution of a large range of biomolecules in tissues. 2,6-Dihydroxyacetophenone (DHA) matrix has been shown to provide efficient ionization of lipids, especially gangliosides. The major drawback for DHA as it applies to MS imaging is that it sublimes under vacuum (low pressure) at the extended time necessary to complete both high spatial and mass resolution MSI studies of whole organs. To overcome the problem of sublimation, we used an atmospheric pressure (AP)-MALDI source to obtain high spatial resolution images of lipids in the brain using a high mass resolution mass spectrometer. Additionally, the advantages of atmospheric pressure and DHA for imaging gangliosides are highlighted. The imaging of [M-H] and [M-HO-H] mass peaks for GD gangliosides showed different distribution, most likely reflecting the different spatial distribution of GD and GD species in the brain. Graphical Abstract ᅟ.

Due to the complexity of proteome samples, only a portion of peptides and thus proteins can be identified in a single LC-MS/MS analysis in current shotgun proteomics methodologies. It has been shown that replicate runs can be used to improve the comprehensiveness of the proteome analysis; however, high-intensity peptides tend to be analyzed repeatedly in different runs, thus reducing the chance of identifying low-intensity peptides. In contrast to commonly used online ESI-MS, offline MALDI decouples the separation from MS acquisition, thus allowing in-depth selection for specific precursor ions. Accordingly, we extended a strategy for offline LC-MALDI MS/MS analysis using a precursor ion exclusion list consisting of all identified peptides in preceding runs. The exclusion list eliminated redundant MS/MS acquisitions in subsequent runs, thus reducing MALDI sample depletion and allowing identification of a larger number of peptide identifications in the cumulative dataset. In the analysis of the digest of an Escherichia coli lysate, the exclusion list strategy resulted in a 25% increase in the number of unique peptide identifications in the second run, in contrast to simply pooling MS/MS data from two replicate runs. To reduce the increased LC analysis time for repeat runs, a four-column multiplexed LC system was developed to carry out separation simultaneously. The multiplexed LC-MALDI MS provides a high-throughput platform to utilize the exclusion list strategy in proteome analysis.

Strong orthogonality between differential ion mobility spectrometry (FAIMS) and mass spectrometry (MS) makes their hybrid a powerful approach to separate isomers and isobars. Harnessing that power depends on high resolution in both dimensions. The ultimate mass resolution and accuracy are delivered by Fourier Transform MS increasingly realized in Orbitrap MS, whereas FAIMS resolution is generally maximized by buffers rich in He or H 2 that elevate ion mobility and lead to prominent non-Blanc effects. However, turbomolecular pumps have lower efficiency for light gas molecules and their flow from the FAIMS stage complicates maintaining the ultra-high vacuum needed for Orbitrap operation. Here we address this challenge via two hardware modifications: (i) a differential pumping step between FAIMS and MS stages and (ii) reconfiguration of vacuum lines to isolate pumping of the Orbitrap ultra-high vacuum region. Either greatly ameliorates the pressure increases upon He or H 2 aspiration. This development enables free optimization of FAIMS carrier gas without concerns about MS performance, maximizing the utility and flexibility of FAIMS/MS platforms. SUPPORTING INFORMATION AVAILABLE: Photos of single and tandem ion funnel interfaces, the plot of backing line pressure with SFI.

Visualizing the differential distribution of carbon–carbon double bond (CC db) positional isomers of unsaturated phospholipids (PL) in tissue sections by use of refined matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) technologies offers a high promise to deeper understand PL metabolism and isomer-specific functions in health and disease. Here we introduce an on-tissue ozonization protocol that enables a particular straightforward derivatization of unsaturated lipids in tissue sections. Collision-induced dissociation (CID) of MALDI-generated ozonide ions (with yields in the several ten percent range) produced the Criegee fragment ion pairs, which are indicative of CC db position(s). We used our technique for visualizing the differential distribution of Δ9 and Δ11 isomers of phosphatidylcholines in mouse brain and in human colon samples with the desorption laser spot size 15 μm, emphasizing the potential of the technique to expose local isomer-specific metabolism of PLs.