Comparison of targeted peptide quantification assays for reductive dehalogenases by selective reaction monitoring (SRM) and precursor reaction monitoring (PRM)

Targeted absolute protein quantification yields valuable information about physiological adaptation of organisms and is thereby of high interest. Especially for this purpose, two proteomic mass spectrometry-based techniques namely selective reaction monitoring (SRM) and precursor reaction monitoring (PRM) are commonly applied. The objective of this study was to establish an optimal quantification assay for proteins with the focus on those involved in housekeeping functions and putative reductive dehalogenase proteins from the strictly anaerobic bacterium Dehalococcoides mccartyi strain CBDB1. This microbe is small and slow-growing; hence, it provides little biomass for comprehensive proteomic analysis. We therefore compared SRM and PRM techniques. Eleven peptides were successfully quantified by both methods. In addition, six peptides were solely quantified by SRM and four by PRM, respectively. Peptides were spiked into a background of Escherichia coli lysate and the majority of peptides were quantifiable down to 500 amol absolute on column by both methods. Peptide quantification in CBDB1 lysate resulted in the detection of 15 peptides using SRM and 14 peptides with the PRM assay. Resulting quantification of five dehalogenases revealed copy numbers of <10 to 115 protein molecules per cell indicating clear differences in abundance of RdhA proteins during growth on hexachlorobenzene. Our results indicated that both methods show comparable sensitivity and that the combination of the mass spectrometry assays resulted in higher peptide coverage and thus more reliable protein quantification.

Resonance-enhanced multiphoton ionization with circularly polarized light: chiral carbonyls

An introduction to the principle and possibilities of the new method of circular dichroism laser mass spectrometry is given and its state of development is reviewed. This method allows enantiosensitive, mass-selective probing of chiral molecules. It is based on the combination of resonance-enhanced multiphoton ionization with circularly polarized light and specially modified time-of-flight mass spectrometry. As an example, application to carbonyls is presented.

Imaging of Noncovalent Complexes by MALDI-MS

Noncovalent interactions govern how molecules communicate. Mass spectrometry is an important and versatile tool for the analysis of noncovalent complexes (NCX). Electrospray mass spectrometry (ESI-MS) is the most widely used MS technique for the study of NCXs because of its softer ionization and easy compatibility with the solution phase of NCX mixtures. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has also been used to study NCXs. However, successful analysis depends upon several experimental factors, such as matrix selection, solution pH, and instrumental parameters. In this study, we employ MALDI imaging mass spectrometry to investigate the location and formation of NCXs, involving both peptides and proteins, in a MALDI sample spot.

Quantification of Saquinavir from Lysates of Peripheral Blood Mononuclear Cells Using Microarrays and Standard MALDI-TOF-MS

Drug monitoring is usually performed by liquid chromatography coupled with optical detection or electrospray ionization mass spectrometry. More recently, matrix-assisted laser desorption/ionization (MALDI) in combination with triple quadrupole or Fourier-transform (FT) mass analyzers has also been reported to allow accurate quantification. Here, we present a strategy that employs standard MALDI time-of-flight (TOF) mass spectrometry (MS) for the sensitive and accurate quantification of saquinavir from an extract of blood peripheral mononuclear cells. Unambiguous identification of saquinavir in the mass spectra was possible because of using internal mass calibration and by an overall low chemical noise in the low mass range. Exact mass determination of the constant background peaks of the cell extract, which were used for recalibration, was performed by an initial MALDI-FT-MS analysis. Fast and multiplexed sample analysis was enabled by microarray technology, which provided 10 replicates in the lower nL range for each sample in parallel lanes on a chip. In order to validate the method, we employed various statistical tests, such as confidence intervals for linear regressions, three quality control samples, and inverse confidence limits of the estimated concentration ratios.

Overcoming Selectivity and Sensitivity Issues of Direct Inject Electrospray Mass Spectrometry via DAPNe-NSI-MS

Direct inject electrospray mass spectrometry offers minimal sample preparation and a “shotgun” approach to analyzing samples. However, complex matrix effects often make direct inject an undesirable sample introduction technique, particularly for trace level analytes. Highlighted here is our solution to the pitfalls of direct inject mass spectrometry and other ambient ionization methods with a focus on trace explosives. Direct analyte-probed nanoextraction coupled to nanospray ionization mass spectrometry solves selectivity issues and reduces matrix effects while maintaining minimal sample preparation requirements. With appropriate solvent conditions, most explosive residues can be analyzed with this technique regardless of the nature of the substance (i.e., nitroaromatic, oxidizing salt, or peroxide).

UPLC-UV-MSE analysis for quantification and identification of major carotenoid and chlorophyll species in algae

A fast method for quantification and identification of carotenoid and chlorophyll species utilizing liquid chromatography coupled with UV detection and mass spectrometry has been demonstrated and validated for the analysis of algae samples. This method allows quantification of targeted pigments and identification of unexpected compounds, providing isomers separation, UV detection, accurate mass measurements, and study of fragment ions for structural elucidation in a single run. This is possible using parallel alternating low- and high-energy collision spectral acquisition modes, which provide accurate mass full scan chromatograms and accurate mass high-energy chromatograms. Here, it is shown how this approach can be used to confirm carotenoid and chlorophyll species by identification of key diagnostic fragmentations during high-energy mode. The developed method was successfully applied for the analysis of Dunaliella salina samples during defined red LED lighting growth conditions, identifying 37 pigments including 19 carotenoid species and 18 chlorophyll species, and providing quantification of 7 targeted compounds. Limit of detections for targeted pigments ranged from 0.01?ng/mL for lutein to 0.24?ng/mL for chlorophyll a. Inter-run precision ranged for of 3 to 24 (RSD%) while inter-run inaccuracy ranged from ?17 to 11.

Measurement and Visualization of Mass Transport for the Flowing Atmospheric Pressure Afterglow (FAPA) Ambient Mass-Spectrometry Source

Ambient desorption/ionization mass spectrometry (ADI-MS) has developed into an important analytical field over the last 9 years. The ability to analyze samples under ambient conditions while retaining the sensitivity and specificity of mass spectrometry has led to numerous applications and a corresponding jump in the popularity of this field. Despite the great potential of ADI-MS, problems remain in the areas of ion identification and quantification. Difficulties with ion identification can be solved through modified instrumentation, including accurate-mass or MS/MS capabilities for analyte identification. More difficult problems include quantification because of the ambient nature of the sampling process. To characterize and improve sample volatilization, ionization, and introduction into the mass spectrometer interface, a method of visualizing mass transport into the mass spectrometer is needed. Schlieren imaging is a well-established technique that renders small changes in refractive index visible. Here, schlieren imaging was used to visualize helium flow from a plasma-based ADI-MS source into a mass spectrometer while ion signals were recorded. Optimal sample positions for melting-point capillary and transmission-mode (stainless steel mesh) introduction were found to be near (within 1 mm of) the mass spectrometer inlet. Additionally, the orientation of the sampled surface plays a significant role. More efficient mass transport resulted for analyte deposits directly facing the MS inlet. Different surfaces (glass slide and rough surface) were also examined; for both it was found that the optimal position is immediately beneath the MS inlet.

High-throughput absolute quantification of proteins using an improved two-dimensional reversed-phase separation and quantification concatemer (QconCAT) approach

Stable isotope dilution–selective reaction monitoring–mass spectrometry (SID-SRM-MS) has been widely used for the absolute quantitative analysis of proteins. However, when performing the large-scale absolute quantification of proteins from a more complex tissue sample, such as mouse liver, in addition to a high-throughput approach for the preparation and calibration of large amounts of stable-isotope-labelled internal standards, a more powerful separation method prior to SRM analysis is also urgently needed. To address these challenges, a high-throughput absolute quantification strategy based on an improved two-dimensional reversed-phase (2D RP) separation and quantification concatemer (QconCAT) approach is presented in this study. This strategy can be used to perform the simultaneous quantification of hundreds of proteins from mouse liver within one week of total MS measurement time. By using calibrated synthesised peptides from the protein glutathione S-transferase (GST), large amounts of GST-tagged QconCAT internal standards corresponding to hundreds of proteins can be accurately and rapidly quantified. Additionally, using an improved 2D RP separation method, a mixture containing a digested sample and QconCAT standards can be efficiently separated and absolutely quantified. When a maximum gradient of 72 min is employed in the first LC dimension, resulting in 72 fractions, identification and absolute quantification experiments for all fractions can be completed within one week of total MS measurement time. The quantification approach developed here can further extend the dynamic range and increase the analytical sensitivity of SRM analysis of complex tissue samples, thereby helping to increase the coverage of absolute quantification in a whole proteome.

Selective Detection of Carbohydrates and Their Peptide Conjugates by ESI-MS Using Synthetic Quaternary Ammonium Salt Derivatives of Phenylboronic Acids

We present new tags based on the derivatives of phenylboronic acid and apply them for the selective detection of sugars and peptide-sugar conjugates in mass spectrometry. We investigated the binding of phenylboronic acid and its quaternary ammonium salt (QAS) derivatives to carbohydrates and peptide-derived Amadori products by HR-MS and MS/MS experiments. The formation of complexes between sugar or sugar-peptide conjugates and synthetic tags was confirmed on the basis of the unique isotopic distribution resulting from the presence of boron atom. Moreover, incorporation of a quaternary ammonium salt dramatically improved the efficiency of ionization in mass spectrometry. It was found that the formation of a complex with phenylboronic acid stabilizes the sugar moiety in glycated peptides, resulting in simplification of the fragmentation pattern of peptide-derived Amadori products. The obtained results suggest that derivatization of phenylboronic acid as QAS is a promising method for sensitive ESI-MS detection of carbohydrates and their conjugates formed by non-enzymatic glycation or glycosylation.

Non-denaturating isoelectric focusing gel electrophoresis for uranium–protein complexes quantitative analysis with LA-ICP MS

A non-denaturating isoelectric focusing (ND-IEF) gel electrophoresis protocol has been developed to study and identify uranium (U)–protein complexes with laser ablation–inductively coupled plasma mass spectrometry (LA-ICP MS) and electrospray ionization mass spectrometry (ESI-MS). The ND-IEF-LA-ICP MS methodology set-up was initiated using in vitro U–protein complex standards (i.e., U–bovine serum albumin and U–transferrin) allowing the assessment of U recovery to 64.4?±?0.4 %. This methodology enabled the quantification of U–protein complexes at 9.03?±?0.23, 15.27?±?0.36, and 177.31?±?25.51 nmol U L^?1 in digestive gland cytosols of the crayfish, Procambarus clarkii, exposed respectively to 0, 0.12, and 2.5 μmol of waterborne depleted U L^?1 during 10 days. ND-IEF-LA-ICP MS limit of detection was 19.3 pmol U L^?1. Elemental ICP MS signals obtained both in ND-IEF electropherograms and in size exclusion chromatograms of in vivo U–protein complexes revealed interactions between U- and Fe- and Cu-proteins. Moreover, three proteins (hemocyanin, pseudohemocyanin-2, and arginine kinase) out of 42 were identified as potential uranium targets in waterborne-exposed crayfish cytosols by microbore reversed phase chromatography coupled to molecular mass spectrometry (µRPC-ESI-MS/MS) after ND-IEF separation.

Protected Amine Labels: A Versatile Molecular Scaffold for Multiplexed Nominal Mass and Sub-Da Isotopologue Quantitative Proteomic Reagents

We assemble a versatile molecular scaffold from simple building blocks to create binary and multiplexed stable isotope reagents for quantitative mass spectrometry. Termed Protected Amine Labels (PAL), these reagents offer multiple analytical figures of merit including, (1) robust targeting of peptide N-termini and lysyl side chains, (2) optimal mass spectrometry ionization efficiency through regeneration of primary amines on labeled peptides, (3) an amino acid-based mass tag that incorporates heavy isotopes of carbon, nitrogen, and oxygen to ensure matched physicochemical and MS/MS fragmentation behavior among labeled peptides, and (4) a molecularly efficient architecture, in which the majority of hetero-atom centers can be used to synthesize a variety of nominal mass and sub-Da isotopologue stable isotope reagents. We demonstrate the performance of these reagents in well-established strategies whereby up to four channels of peptide isotopomers, each separated by 4 Da, are quantified in MS-level scans with accuracies comparable to current commercial reagents. In addition, we utilize the PAL scaffold to create isotopologue reagents in which labeled peptide analogs differ in mass based on the binding energy in carbon and nitrogen nuclei, thereby allowing quantification based on MS or MS/MS spectra. We demonstrate accurate quantification for reagents that support 6-plex labeling and propose extension of this scheme to 9-channels based on a similar PAL scaffold. Finally, we provide exemplar data that extend the application of isotopologe-based quantification reagents to medium resolution, quadrupole time-of-flight mass spectrometers.

Screening of Complicated Matrixes with Paper Assisted Ultrasonic Spray Ionization Mass Spectrometry

To analyze compounds in complicated matrixes using mass spectrometry, we describe a novel ambient ionization approach, termed paper assisted ultrasonic spray ionization (PAUSI). The ionization process is based on the ultrasonic vibration of the piezoelectric ceramic disk, on which the samples are placed. Porous materials are utilized to generate fine initial droplet, which could alleviate matrix effect during ionization process for complicated matrix. PAUSI was evaluated as an attractive tool to screen analytes from complicated matrixes, such as (1) bovine serum with NaCl 150 g/L, (2) viscous samples, and (3) biological fluid, without any sample preparation. Moreover, it provides great advantage in simplifying the mass spectrometry analysis process, and the ionization device is inexpensive and easy to operate.

Analytical Properties of Solid-substrate Electrospray Ionization Mass Spectrometry

Conventional electrospray ionization mass spectrometry (ESI-MS) uses a capillary for sample loading and ionization. Along with the development of ambient ionization techniques, ESI-MS using noncapillary emitters has attracted more interest in recent years. Following our recent report on ESI-MS using wooden tips (Anal. Chem. 83, 8201–8207 (2011)), the technique was further investigated and extended in this study. Our results revealed that the wooden tips could serve as a chromatographic column for separation of sample components. Sequential and exhaustive ionization was observed for proteins and salts on wooden tips with salts ionized sooner and proteins later. Nonconductive materials that contain microchannels/pores could be used as tips for ESI-MS analysis with sample solutions loaded to the sharp-ends only, since rapid diffusion of sample solutions by capillary action would enable the tips to become conductive. Tips of inert materials such as bamboo, fabrics, and sponge could be used for sample loading and ionization, while samples such as tissue, mushroom, and bone could form tips to induce ionization for direct analysis with application of a high voltage.

Toward high spatial resolution sampling and characterization of biological tissue surfaces using mass spectrometry

Mass spectrometry has emerged as a powerful tool for the bioanalytical sciences because of its ability to characterize small and large biomolecules in vanishingly small amounts. A recurring motif in mass spectrometry aims to decipher the chemical composition of biological samples at the molecular level, requiring drastic improvements in the ability to interrogate well defined and highly spatially resolved areas of a sample surface. With the growth of novel ionization methods, numerous advances have been made in sampling biological tissue surfaces. Here, current advancements in ambient, inlet, and vacuum ionization methods are discussed with respect to the potential improvements in the goal of achieving high spatial resolution and/or fast surface analysis. Of similar importance is the need for improvements in applicable characterization strategies using high performance fragmentation technologies such as electron transfer dissociation and electron capture dissociation directly from surfaces, and gas-phase separation through ion mobility spectrometry and high resolution mass spectrometry.

Gas chromatography/multiphoton ionization/time-of-flight mass spectrometry using a femtosecond laser

A laser can be used for the selective excitation and subsequent ionization of a molecule with an absorption band at the laser wavelength. This technique of multiphoton ionization (MPI), when combined with time-of-flight mass spectrometry (TOF-MS), permits the efficient detection of induced ions in mass analysis. This combination of MPI/TOF-MS can be coupled with gas chromatography (GC) to achieve even more enhanced selectivity. Thus, GC/MPI/TOF-MS can be employed for trace analysis of samples containing numerous chemical species. A variety of laser sources have been used for this purpose. Since molecules that are classified as environmental pollutants frequently contain chlorine and bromine atoms, the lifetime of the excited state can be decreased by energy transfer from the singlet to triplet levels by spin–orbit interaction. A high-power femtosecond laser with a pulse width shorter than the lifetime of the analyte molecule provides femtogram or even subfemtogram detection limits, which have not yet been achieved using the most sensitive high-resolution double-focus sector-type mass spectrometers. Numerous environmental pollutants such as dioxins in soils and pesticides in foods have been successfully quantified using GC/MPI/TOF-MS, and this technique has proven itself to be a useful and practical method for trace analysis.

Using electrospray laser desorption ionization mass spectrometry to rapidly examine the integrity of proteins stored in various solutions

Electrospray laser desorption ionization mass spectrometry (ELDI/MS) allows the rapid desorption and ionization of proteins from solutions under ambient conditions. In this study, we have demonstrated the use of ELDI/MS to efficiently examine the integrity of the proteins stored in various solutions before they were further used for other biochemical tests. The protein standards were prepared in the solutions containing buffers, organic salts, inorganic salts, strong acid, strong base, and organic solvents, respectively, to simulate those collected from solvent extraction, filtration, dialysis, or chromatographic separation. Other than the deposit of a drop of the sample solution on the metallic sample plate in an ELDI source, no additional sample pretreatment is needed. The sample drop was then irradiated with a pulsed laser; this led to desorption of the analyte molecules, which subsequently entered the ESI plume to undergo post-ionization. Because adjustment of the composition of the sample solution is unnecessary, this technique appears to be useful for rapidly evaluating the integrity of proteins after storage or prior to further biochemical treatment. In addition, when using acid-free and low-organic-solvent ESI solutions for ELDI/MS analysis, the native conformations of the proteins in solution could be detected.

Mass Spectrometric Imaging of Red Fluorescent Protein in Breast Tumor Xenografts

Mass spectrometric imaging (MSI) in combination with electrospray mass spectrometry (ESI-MS) is a powerful technique for visualization and identification of a variety of different biomolecules directly from thin tissue sections. As commonly used tools for molecular reporting, fluorescent proteins are molecular reporter tools that have enabled the elucidation of a multitude of biological pathways and processes. To combine these two approaches, we have performed targeted MS analysis and MALDI-MSI visualization of a tandem dimer (td)Tomato red fluorescent protein, which was expressed exclusively in the hypoxic regions of a breast tumor xenograft model. For the first time, a fluorescent protein has been visualized by both optical microscopy and MALDI-MSI. Visualization of tdTomato by MALDI-MSI directly from breast tumor tissue sections will allow us to simultaneously detect and subsequently identify novel molecules present in hypoxic regions of the tumor. MS and MALDI-MSI of fluorescent proteins, as exemplified in our study, is useful for studies in which the advantages of MS and MSI will benefit from the combination with molecular approaches that use fluorescent proteins as reporters.

Ambient DESI and LESA-MS Analysis of Proteins Adsorbed to a Biomaterial Surface Using In-Situ Surface Tryptic Digestion

The detection and identification of proteins adsorbed onto biomaterial surfaces under ambient conditions has significant experimental advantages but has proven to be difficult to achieve with conventional measuring technologies. In this study, we present an adaptation of desorption electrospray ionization (DESI) and liquid extraction surface analysis (LESA) mass spectrometry (MS) coupled with in-situ surface tryptic digestion to identify protein species from a biomaterial surface. Cytochrome c, myoglobin, and BSA in a combination of single and mixture spots were printed in an array format onto Permanox slides, followed by in-situ surface digestion and detection via MS. Automated tandem MS performed on surface peptides was able to identify the proteins via MASCOT. Limits of detection were determined for DESI-MS and a comparison of DESI and LESA-MS peptide spectra characteristics and sensitivity was made. DESI-MS images of the arrays were produced and analyzed with imaging multivariate analysis to automatically separate peptide peaks for each of the proteins within a mixture into distinct components. This is the first time that DESI and LESA-MS have been used for the in-situ detection of surface digested proteins on biomaterial surfaces and presents a promising proof of concept for the use of ambient MS in the rapid and automated analysis of surface proteins.

Dried Blood Spot Proteomics: Surface Extraction of Endogenous Proteins Coupled with Automated Sample Preparation and Mass Spectrometry Analysis

Dried blood spots offer many advantages as a sample format including ease and safety of transport and handling. To date, the majority of mass spectrometry analyses of dried blood spots have focused on small molecules or hemoglobin. However, dried blood spots are a potentially rich source of protein biomarkers, an area that has been overlooked. To address this issue, we have applied an untargeted bottom-up proteomics approach to the analysis of dried blood spots. We present an automated and integrated method for extraction of endogenous proteins from the surface of dried blood spots and sample preparation via trypsin digestion by use of the Advion Biosciences Triversa Nanomate robotic platform. Liquid chromatography tandem mass spectrometry of the resulting digests enabled identification of 120 proteins from a single dried blood spot. The proteins identified cross a concentration range of four orders of magnitude. The method is evaluated and the results discussed in terms of the proteins identified and their potential use as biomarkers in screening programs.

Selective and Nonselective Cleavages in Positive and Negative CID of the Fragments Generated from In-Source Decay of Intact Proteins in MALDI-MS

Selective and nonselective cleavages in ion trap low-energy collision-induced dissociation (CID) experiments of the fragments generated from in-source decay (ISD) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) of intact proteins are described in both positive and negative ion modes. The MALDI-ISD spectra of the proteins demonstrate common, discontinuous, abundant c- and z′-ions originating from cleavage at the N–Cα bond of Xxx-Asp/Asn and Gly-Xxx residues in both positive- and negative-ion modes. The positive ion CID of the c- and z′-ions resulted in product ions originating from selective cleavage at Asp-Xxx, Glu-Xxx and Cys-Xxx residues. Nonselective cleavage product ions rationalized by the mechanism of a “mobile proton” are also observed in positive ion CID spectra. Negative ion CID of the ISD fragments results in complex product ions accompanied by the loss of neutrals from b-, c-, and y-ions. The most characteristic feature of negative ion CID is selective cleavage of the peptide bonds of acidic residues, Xxx-Asp/Glu/Cys. A definite influence of α-helix on the CID product ions was not obtained. However, the results from positive ion and negative ion CID of the MALDI-ISD fragments that may have long α-helical domains suggest that acidic residues in helix-free regions tend to degrade more than those in helical regions.