Evaluation of 6 MALDI-Matrices for 10 μm Lipid Imaging and On-Tissue MSn with AP-MALDI-Orbitrap.

Mass spectrometry imaging is a technique uniquely suited to localize and identify lipids in a tissue sample. Using an atmospheric pressure (AP-) matrix-assisted laser desorption ionization (MALDI) source coupled to an Orbitrap Elite, numerous lipid locations and structures can be determined in high mass resolution spectra and at cellular spatial resolution, but careful sample preparation is necessary. We tested 11 protocols on serial brain sections for the commonly used MALDI matrices CHCA, norharmane, DHB, DHAP, THAP, and DAN in combination with tissue washing and matrix additives to determine the lipid coverage, signal intensity, and spatial resolution achievable with AP-MALDI.

Rapid and Quantitative Analysis of Aflatoxin M1 From Milk Using Atmospheric Pressure-Matrix Assisted Laser Desorption/Ionization (AP-MALDI)-Triple Quadrupole Selected Reaction Monitoring.

Background: Aflatoxin M1 (AFM1) is a carcinogenic hydroxylated metabolite commonly found in milk. It is relatively stable toward decontamination procedures posing a major health risk, and it requires an international regulatory mandate of detection at trace levels.

Objective: To develop a high-throughput, reliable, and compliant method for the identification of AFM1 in milk samples using atmospheric pressure-matrix assisted laser desorption/ionization (AP-MALDI) selected reaction monitoring (SRM) quantitation.

Rapid Detection of Viral Envelope Lipids Using Lithium Adducts and AP-MALDI High-Resolution Mass Spectrometry.

There is an unmet need to develop analytical strategies that not only characterize the lipid composition of the viral envelope but also do so on a time scale that would allow for high-throughput analysis. With that in mind, we report the use of atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) high-resolution mass spectrometry (HRMS) combined with lithium adduct consolidation to profile total lipid extracts rapidly and confidently from enveloped viruses. The use of AP-MALDI reduced the dependency of using a dedicated MALDI mass spectrometer and allowed for interfacing the MALDI source to a mass spectrometer with the desired features, which included high mass resolving power (>100000) and tandem mass spectrometry.

Ozonization of Tissue Sections for MALDI MS Imaging of Carbon-Carbon Double Bond Positional Isomers of Phospholipids.

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).

High-Resolution Differential Ion Mobility Separations/Orbitrap Mass Spectrometry without Buffer Gas Limitations.

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 that elevate ion mobility and lead to prominent non-Blanc effects.

MALDI MS Imaging at Acquisition Rates Exceeding 100 Pixels per Second.

The practicality of matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) applied to molecular imaging of biological tissues is limited by the analysis speed. Typically, a relatively low speed of stop-and-go micromotion of XY stages is considered as a factor substantially reducing the rate with which fresh sample material can be supplied to the laser spot. The sample scan rate in our laboratory-built high-throughput imaging TOF mass spectrometer was significantly improved through the use of a galvanometer-based optical scanner performing fast laser spot repositioning on a target plate.

Characterizing and alleviating ion suppression effects in atmospheric pressure matrix-assisted laser desorption/ionization.

Rationale: As a powerful ambient ion source, atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) enables direct analysis at atmospheric pressure/temperature and minimal sample preparation. With the increasing usage of AP-MALDI sources with Orbitrap instruments, systematic characterization of the extent of ion suppression effect (ISE) in AP-MALDI-Orbitrap mass spectrometry imaging (MSI) is desirable. Recently, a new low-pressure MALDI platform has been introduced that reportedly provided better sensitivity.

AP-MALDI Mass Spectrometry Imaging of Gangliosides Using 2,6-Dihydroxyacetophenone.

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.

A comparative study on the analytical utility of atmospheric and low-pressure MALDI sources for the mass spectrometric characterization of peptides.

A comparative MS study was conducted on the analytical performance of two matrix-assisted laser desorption/ionization (MALDI) sources that operated at either low pressure (∼1Torr) or at atmospheric pressure. In both cases, the MALDI sources were attached to a linear ion trap mass spectrometer equipped with a two-stage ion funnel. The obtained results indicate that the limits of detection, in the analysis of identical peptide samples, were much lower with the source that was operated slightly below the 1-Torr pressure.

Comparative studies of peak intensities and chromatographic separation of proteolytic digests, PTMs, and intact proteins obtained by nanoLC-ESI MS analysis at room and elevated temperatures.

This work demonstrates that the chromatographic separation performed at highly stabilized elevated temperature results in significant improvements in sensitivity, quantitative accuracy, chromatographic resolution, and run-to-run reproducibility of nanoLC-MS analysis of complex peptides mixtures. A newly developed platform was shown to provide conditions for accurate temperature stabilization and temperature homogeneity when performing nanoLC-ESI MS analysis. We quantitatively assessed and compared the recovery of peptides and small proteins from nanoLC columns at room and elevated temperatures.

Numerical analysis of ion-funnel transmission efficiency in an API-MS system with a continuum/microscopic approach.

A multi-step numerical approach is used to analyze the efficiency of an ion-funnel to transport ions over a wide range of m/z. A continuum approach based on the solution of the Navier-Stokes equations is applied to model the gas flow through a capillary connecting the atmospheric and subatmospheric sections of a mass spectrometer. A microscopic, fully kinetic approach based on the solution of the Boltzmann equation is used to examine the ion and gas transport through an ion-funnel kept at a 0.

Ghost peaks observed after atmospheric pressure matrix-assisted laser desorption/ionization experiments may disclose new ionization mechanism of matrix-assisted hypersonic velocity impact ionization.

Rationale: Understanding the mechanisms of matrix-assisted laser desorption/ionization (MALDI) promises improvements in the sensitivity and specificity of many established applications in the field of mass spectrometry. This paper reports a serendipitous observation of a significant ion yield in a post-ionization experiment conducted after the sample had been removed from a standard atmospheric pressure (AP)-MALDI source. This post-ionization is interpreted in terms of collisions of microparticles moving with a hypersonic velocity into a solid surface.

Numerical modeling of ion transport in an ESI-MS system.

Gas and ion transport in the capillary-skimmer subatmospheric interface of a mass spectrometer, which is typically utilized to separate unevaporated micro-droplets from ions, was studied numerically using a two-step approach spanning multiple gas dynamic regimes. The gas flow in the heated capillary and in the interface was determined by solving numerically the Navier-Stokes equation. The capillary-to-skimmer gas/ion flow was modeled through the solution of the full Boltzmann equation with a force term.

Rapid matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging with scanning desorption laser beam.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) imaging of surfaces and tissues is a rapidly evolving technique having great potential in the field of biosciences. In earlier times, acquisition of a single high-resolution MS image could take days. Despite the recent introduction of high-repetition rate lasers to increase sample throughput of axial TOF MS instruments, obtaining a high-resolution image still requires a few hours.

Comparison of in-gel protein separation techniques commonly used for fractionation in mass spectrometry-based proteomic profiling.

Fractionation of complex samples at the cellular, subcellular, protein, or peptide level is an indispensable strategy to improve the sensitivity in mass spectrometry-based proteomic profiling. This study revisits, evaluates, and compares the most common gel-based protein separation techniques i.e.

Limitation of predictive 2-D liquid chromatography in reducing the database search space in shotgun proteomics: in silico studies.

A two-dimensional (2-D) liquid chromatography (LC) separation of complex peptide mixtures that combines a normal phase utilizing hydrophilic interactions and a reversed phase offers reportedly the highest level of 2-D LC orthogonality by providing an even spread of peptides across multiple LC fractions. Matching experimental peptide retention times to those predicted by empirical models describing chromatographic separation in each LC dimension leads to a significant reduction in a database search space. In this work, we calculated the retention times of tryptic peptides separated in the C18 reversed phase at different separation conditions (pH 2 and pH 10) and in TSK gel Amide-80 normal phase.

Sequence-specific predictive chromatography to assist mass spectrometric analysis of asparagine deamidation and aspartate isomerization in peptides.

Deamidation of asparagine and spontaneous isomerization of aspartic acid in proteins and peptides occur frequently. These modifications result in a mixture of peptide variants containing all three residues in the sequences. Identification and isomer quantification for these systems are challenging tasks for tandem mass spectrometry commonly utilized in protein analysis.

Retention time prediction using the model of liquid chromatography of biomacromolecules at critical conditions in LC-MS phosphopeptide analysis.

LC combined with MS/MS analysis of complex mixtures of protein digests is a reliable and sensitive method for characterization of protein phosphorylation. Peptide retention times (RTs) measured during an LC-MS/MS run depend on both the peptide sequence and the location of modified amino acids. These RTs can be predicted using the LC of biomacromolecules at critical conditions model (BioLCCC).

On the high-resolution mass analysis of the product ions in tandem time-of-flight (TOF/TOF) mass spectrometers using a time-dependent re-acceleration technique.

The time-dependent reacceleration of product ions produced as a result of dissociation of a single precursor ion in a tandem time-of-flight mass spectrometer is considered for the first time. Analytical expressions for the shapes of electric pulses bringing all the kinetic energies of the product ions to the same value are derived for two cases: forward acceleration mode and deceleration, followed by re-acceleration in the reversed direction (reversed mode). Secondary time-of-flight focusing resulting from the re-acceleration in the reversed mode is shown to be mass-dependent and, when averaged over a wide mass range, the focusing is tight enough to provide mass resolution exceeding 10,000.

Comparative performance of tripole and quadrupole ion guides at elevated pressure.

This study presents the first practical demonstration of an operational tripole ion guide. The transmission was measured for both the tripole and quadrupole ion guides at 1 Torr pressure. It was found that the quadrupole provides 2.

Enhanced characterization of complex proteomic samples using LC-MALDI MS/MS: exclusion of redundant peptides from MS/MS analysis in replicate runs.

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.

High-speed, high-resolution monolithic capillary LC-MALDI MS using an off-line continuous deposition interface for proteomic analysis.

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.

Coumarin tags for analysis of peptides by MALDI-TOF MS and MS/MS. 2. Alexa Fluor 350 tag for increased peptide and protein Identification by LC-MALDI-TOF/TOF MS.

The goal of this study was the development of N-terminal tags to improve peptide identification using high-throughput MALDI-TOF/TOF MS. Part 1 of the study was focused on the influence of derivatization on the intensities of MALDI-TOF MS signals of peptides. In part 2, various derivatization approaches for the improvement of peptide fragmentation efficiency in MALDI-TOF/TOF MS are explored.

Increased identification of peptides by enhanced data processing of high-resolution MALDI TOF/TOF mass spectra prior to database searching.

This paper presents application of sequential enhanced data processing procedures to high-resolution tandem mass spectra for identification of peptides using the Mascot database search algorithm. A strategy for (1) selection of fragment ion peaks from MS/MS spectra, (2) utilization of improved mass accuracy of the precursor ions, and (3) wavelet denoising of the mass spectra prior to fragment ion selection have been developed. The number of peptide identifications obtained using the enhanced processing was then compared with that obtained using software provided by the instrument manufacturer.

Coumarin tags for improved analysis of peptides by MALDI-TOF MS and MS/MS. 1. Enhancement in MALDI MS signal intensities.

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.