Challenges in mass spectrometry-based quantification of bioactive peptides: A case study exploring the neuropeptide Y family

The study of biologically active peptides is critical to the understanding of physiological pathways, especially those involved in the development of disease. Historically, the measurement of biologically active endogenous peptides has been undertaken by radioimmunoassay, a highly sensitive and robust technique that permits the detection of physiological concentrations in different biofluid and tissue extracts. Over recent years, a range of mass spectrometric approaches have been applied to peptide quantification with limited degrees of success. Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) belong to the NPY family exhibiting regulatory effects on appetite and feeding behavior. The physiological significance of these peptides depends on their molecular forms and in vivo concentrations systemically and at local sites within tissues. In this report, we describe an approach for quantification of individual peptides within mixtures using high-performance liquid chromatography electrospray ionization tandem mass spectrometry analysis of the NPY family peptides. Aspects of quantification including sample preparation, the use of matrix-matched calibration curves, and internal standards will be discussed. This method for the simultaneous determination of NPY, PYY, and PP was accurate and reproducible but lacks the sensitivity required for measurement of their endogenous concentration in plasma. The advantages of mass spectrometric quantification will be discussed alongside the current obstacles and challenges. © 2012 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98: 357–366, 2012.

Preliminary results on the characterisation of organic aerosols in urban schools by Aerosol Mass Spectrometry

Vehicle emissions have been linked to detrimental health effects with children thought to be more susceptible (See e.g., Ryan et al 2005). In an urban environment a major source of organic aerosols (OA) are vehicle emissions. The ambient concentration of OA is dynamic in nature and the use of an aerosol mass spectrometer can achieve the necessary temporal resolution to capture the daily variation of OA (Jimenez et al 2009). Currently there is a limited understanding of effects of long term exposure to traffic emissions on children’s health. In the present study, we used an aerosol mass spectrometer to monitor OA and determine children’s potential exposure at school to traffic emissions.In this paper, we present the preliminary results of this investigation. The study is a part of a larger project aimed at gaining a holistic picture of the exposure of children to traffic related pollutants, known as UPTECH (www.ilaqh.qut.edu.au/Misc/ UPTECH%20Home.htm).

Mass spectrometry-based metabolomics towards understanding of gene functions with a diversity of biological contexts

Currently, mass spectrometry-based metabolomics studies extend beyond conventional chemical categorization and metabolic phenotype analysis to understanding gene function in various biological contexts (e.g., mammalian, plant, and microbial). These novel utilities have led to many innovative discoveries in the following areas: disease pathogenesis, therapeutic pathway or target identification, the biochemistry of animal and plant physiological and pathological activities in response to diverse stimuli, and molecular signatures of host-pathogen interactions during microbial infection. In this review, we critically evaluate the representative applications of mass spectrometry-based metabolomics to better understand gene function in diverse biological contexts, with special emphasis on working principles, study protocols, and possible future development of this technique. Collectively, this review raises awareness within the biomedical community of the scientific value and applicability of mass spectrometry-based metabolomics strategies to better understand gene function, thus advancing this application's utility in a broad range of biological fields

Analysis of organic aerosols collected on filters by Aerosol Mass Spectrometry for source identification

Aerosol mass spectrometers (AMS) are powerful tools in the analysis of the chemical composition of airborne particles, particularly organic aerosols which are gaining increasing attention. However, the advantages of AMS in providing on-line data can be outweighed by the difficulties involved in its use in field measurements at multiple sites. In contrast to the on-line measurement by AMS, a method which involves sample collection on filters followed by subsequent analysis by AMS could significantly broaden the scope of AMS application. We report the application of such an approach to field studies at multiple sites. An AMS was deployed at 5 urban schools to determine the sources of the organic aerosols at the schools directly. PM1 aerosols were also collected on filters at these and 20 other urban schools. The filters were extracted with water and the extract run through a nebulizer to generate the aerosols, which were analysed by an AMS. The mass spectra from the samples collected on filters at the 5 schools were found to have excellent correlations with those obtained directly by AMS, with r2 ranging from 0.89 to 0.98. Filter recoveries varied between the schools from 40 -115%, possibly indicating that this method provides qualitative rather than quantitative information. The stability of the organic aerosols on Teflon filters was demonstrated by analysing samples stored for up to two years. Application of the procedure to the remaining 20 schools showed that secondary organic aerosols were the main source of aerosols at the majority of the schools. Overall, this procedure provides accurate representation of the mass spectra of ambient organic aerosols and could facilitate rapid data acquisition at multiple sites where AMS could not be deployed for logistical reasons.

Advances in mass spectrometry for lipidomics

Recent expansion in research in the field of lipidomics has been driven by the development of new mass spectrometric tools and protocols for the identification and quantification of molecular lipids in complex matrices. Although there are similarities between the field of lipidomics and the allied field of mass spectrometry (e.g., proteomics), lipids present some unique advantages and challenges for mass spectrometric analysis. The application of electrospray ionization to crude lipid extracts without prior fractionation-the so-called shotgun approach-is one such example, as it has perhaps been more successfully applied in lipidomics than in any other discipline. Conversely, the diverse molecular structure of lipids means that collision-induced dissociation alone may be limited in providing unique descriptions of complex lipid structures, and the development of additional, complementary tools for ion activation and analysis is required to overcome these challenges. In this article, we discuss the state of the art in lipid mass spectrometry and highlight several areas in which current approaches are deficient and further innovation is required.

Surface analysis of lipids by mass spectrometry : More than just imaging

Mass spectrometry is now an indispensable tool for lipid analysis and is arguably the driving force in the renaissance of lipid research. In its various forms, mass spectrometry is uniquely capable of resolving the extensive compositional and structural diversity of lipids in biological systems. Furthermore, it provides the ability to accurately quantify molecular-level changes in lipid populations associated with changes in metabolism and environment; bringing lipid science to the "omics" age. The recent explosion of mass spectrometry-based surface analysis techniques is fuelling further expansion of the lipidomics field. This is evidenced by the numerous papers published on the subject of mass spectrometric imaging of lipids in recent years. While imaging mass spectrometry provides new and exciting possibilities, it is but one of the many opportunities direct surface analysis offers the lipid researcher. In this review we describe the current state-of-the-art in the direct surface analysis of lipids with a focus on tissue sections, intact cells and thin-layer chromatography substrates. The suitability of these different approaches towards analysis of the major lipid classes along with their current and potential applications in the field of lipid analysis are evaluated. © 2013 Elsevier Ltd. All rights reserved.

Paint spray mass spectrometry for the detection of additives from polymers on conducting surfaces

Paint Spray is developed as a direct sampling ionisation method for mass spectrometric analysis of additives in polymer-based surface coatings. The technique simply involves applying an external high voltage (5 kV) to the wetted sample placed in front of the mass spectrometer inlet and represents a much simpler ionisation technique compared to those currently available. The capabilities of Paint Spray are demonstrated herein with the detection of four commercially available hindered amine light stabilisers; TINUVIN® 770, TINUVIN® 292, TINUVIN® 123 and TINUVIN® 152 directly from thermoset polyester-based coil coatings. Paint Spray requires no sample preparation or pre-treatment and combined with its simplicity - requiring no specialised equipment - makes it ideal for use by non-specialists. The application of Paint Spray for industrial use has significant potential as sample collection from a coil coating production line and Paint Spray ionisation could enable fast quality control screening at high sensitivity.

Characterising in situ activation and degradation of hindered amine light stabilisers using liquid extraction surface analysis-mass spectrometry

Changes in the molecular structure of polymer antioxidants such as hindered amine light stabilisers (HALS) is central to their efficacy in retarding polymer degradation and therefore requires careful monitoring during their in-service lifetime. The HALS, bis-(1-octyloxy-2,2,6,6-tetramethyl-4-piperidinyl) sebacate (TIN123) and bis-(1,2,2,6,6-pentamethyl-4-piperidinyl) sebacate (TIN292), were formulated in different polymer systems and then exposed to various curing and ageing treatments to simulate in-service use. Samples of these coatings were then analysed directly using liquid extraction surface analysis (LESA) coupled with a triple quadrupole mass spectrometer. Analysis of TIN123 formulated in a cross-linked polyester revealed that the polymer matrix protected TIN123 from undergoing extensive thermal degradation that would normally occur at 292 degrees C, specifically, changes at the 1- and 4-positions of the piperidine groups. The effect of thermal versus photo-oxidative degradation was also compared for TIN292 formulated in polyacrylate films by monitoring the in situ conversion of N-CH3 substituted piperidines to N-H. The analysis confirmed that UV light was required for the conversion of N-CH3 moieties to N-H - a major pathway in the antioxidant protection of polymers - whereas this conversion was not observed with thermal degradation. The use of tandem mass spectrometric techniques, including precursor-ion scanning, is shown to be highly sensitive and specific for detecting molecular-level changes in HALS compounds and, when coupled with LESA, able to monitor these changes in situ with speed and reproducibility. (C) 2013 Elsevier B. V. All rights reserved.

Structural characterization of glycerophospholipids by combinations of ozone- and collision-induced dissociation mass spectrometry: The next step towards "top-down" lipidomics

The complete structural elucidation of complex lipids, including glycerophospholipids, using only mass spectrometry represents a major challenge to contemporary analytical technologies. Here, we demonstrate that product ions arising from the collision-induced dissociation (CID) of the [M + Na] + adduct ions of phospholipids can be isolated and subjected to subsequent gas-phase ozonolysis-known as ozone-induced dissociation (OzID)-in a linear ion-trap mass spectrometer. The resulting CID/OzID experiment yields abundant product ions that are characteristic of the acyl substitution on the glycerol backbone (i.e., sn-position). This approach is shown to differentiate sn-positional isomers, such as the regioisomeric phosphatidylcholine pair of PC 16:0/18:1 and PC 18:1/16:0. Importantly, CID/OzID provides a sensitive diagnostic for the existence of an isomeric mixture in a given sample. This is of very high value for the analysis of tissue extracts since CID/OzID analyses can reveal changes in the relative abundance of isomeric constituents even within different tissues from the same animal. Finally, we demonstrate the ability to assign carbon-carbon double bond positions to individual acyl chains at specific backbone positions by adding subsequent CID and/or OzID steps to the workflow and that this can be achieved in a single step using a hybrid triple quadrupole-linear ion trap mass spectrometer. This unique approach represents the most complete and specific structural analysis of lipids by mass spectrometry demonstrated to date and is a significant step towards comprehensive top-down lipidomics. This journal is © The Royal Society of Chemistry 2014. Grant Number ARC/DP0986628, ARC/FT110100249, ARC/LP110200648

Rapid differentiation of isomeric lipids by photodissociation mass spectrometry of fatty acid derivatives

RATIONALE Both traditional electron ionization and electrospray ionization tandem mass spectrometry have demonstrated limitations in the unambiguous identification of fatty acids. In the former case, high electron energies lead to extensive dissociation of the radical cations from which little specific structural information can be obtained. In the latter, conventional collision-induced dissociation (CID) of even-electron ions provides little intra-chain fragmentation and thus few structural diagnostics. New approaches that harness the desirable features of both methods, namely radical-driven dissociation with discrete energy deposition, are thus required. METHODS Herein we describe the derivatization of a structurally diverse suite of fatty acids as 4-iodobenzyl esters (FAIBE). Electrospray ionization of these derivatives in the presence of sodium acetate yields abundant [M+Na]+ ions that can be mass-selected and subjected to laser irradiation (=266nm) on a modified linear ion-trap mass spectrometer. RESULTS Photodissociation (PD) of the FAIBE derivatives yields abundant radical cations by loss of atomic iodine and in several cases selective dissociation of activated carboncarbon bonds (e.g., at allylic positions) are also observed. Subsequent CID of the [M+NaI]center dot+ radical cations yields radical-directed dissociation (RDD) mass spectra that reveal extensive carboncarbon bond dissociation without scrambling of molecular information. CONCLUSIONS Both PD and RDD spectra obtained from derivatized fatty acids provide a wealth of structural information including the position(s) of unsaturation, chain-branching and hydroxylation. The structural information obtained by this approach, in particular the ability to rapidly differentiate isomeric lipids, represents a useful addition to the lipidomics tool box. Copyright (c) 2013 John Wiley & Sons, Ltd.

Identification of abundant alkyl ether glycerophospholipids in the human lens by tandem mass spectrometry techniques

Previous studies have shown that the human lens contains glycerophospholipids with ether linkages. These lipids differ from conventional glycerophospholipids in that the sn-1 substituent is attached to the glycerol backbone via an 1-O-alkyl or an 1-O-alk-1'-enyl ether rather than an ester bond. The present investigation employed a combination of collision-induced dissociation (CID) and ozone-induced dissociation (OzID) to unambiguously distinguish such 1-O-alkyl and 1-O-alk-1'-enyl ethers. Using these methodologies the human lens was found to contain several abundant 1-O-alkyl glycerophos-phoethanolamines, including GPEtn(16:0e/9Z-18:1), GPEtn(11Z-18:1e/9Z-18:1), and GPEtn(18:0e/9Z-18:1), as well as a related series of unusual 1-O-alkyl glycerophosphoserines, including GPSer(16:0e/9Z-18:1), GPSer(11Z-18:1e/9Z-18:1), GPSer(18:0e/9Z-18:1) that to our knowledge have not previously been observed in human tissue. Isomeric 1-O-alk-1'-enyl ethers were absent or in low abundance. Examination of the double bond position within the phospholipids using OzID revealed that several positional isomers were present, including sites of unsaturation at the n-9, n-7, and even n-5 positions. Tandem CID/OzID experiments revealed a preference for double bonds in the n-7 position of 1-O-ether linked chains, while n-9 double bonds predominated in the ester-linked fatty acids [e.g., GPEtn(11Z-18:1e/9Z-18:1) and GPSer(11Z-18:1e/9Z-18:1)]. Different combinations of these double bond positional isomers within chains at the sn-1 and sn-2 positions point to a remarkable molecular diversity of ether-lipids within the human lens.

Differentiation of complex lipid isomers by radical-directed dissociation mass spectrometry

Contemporary lipidomics protocols are dependent on conventional tandem mass spectrometry for lipid identification. This approach is extremely powerful for determining lipid class and identifying the number of carbons and the degree of unsaturation of any acyl-chain substituents. Such analyses are however, blind to isomeric variants arising from different carbon carbon bonding motifs within these chains including double bond position, chain branching, and cyclic structures. This limitation arises from the fact that conventional, low energy collision-induced dissociation of even-electron lipid ions does not give rise to product ions from intrachain fragmentation of the fatty acyl moieties. To overcome this limitation, we have applied radical-directed dissociation (RDD) to the study of lipids for the first time. In this approach, bifunctional molecules that contain a photocaged radical initiator and a lipid-adducting group, such as 4-iodoaniline and 4-iodobenzoic acid, are used to form noncovalent complexes (i.e., adduct ions) with a lipid during electrospray ionization. Laser irradiation of these complexes at UV wavelengths (266 nm) cleaves the carbon iodine bond to liberate a highly reactive phenyl radical. Subsequent activation of the nascent radical ions results in RDD with significant intrachain fragmentation of acyl moieties. This approach provides diagnostic fragments that are associated with the double bond position and the positions of chain branching in glycerophospholipids, sphingomyelins and triacylglycerols and thus can be used to differentiate isomeric lipids differing only in such motifs. RDD is demonstrated for well-defined lipid standards and also reveals lipid structural diversity in olive oil and human very-low density lipoprotein.

Tandem mass spectrometry of deprotonated iodothyronines

In order to assist with the development of more selective and sensitive methods for thyroid hormone analysis the \[M-H](-) anions of the iodothyronines T4, T3, rT3, (3,5)-T2 and the non-iodinated thyronine (TO) have been generated by negative ion electrospray mass spectrometry. Tandem mass spectra of these ions were recorded on a triple-quadrupole mass spectrometer and show a strong analogy with the fragmentation pathways of the parent compound, tyrosine. All iodothyronines also show significant abundances of the iodide anion in their tandem mass spectra, which represents an attractive target for multiple reaction monitoring (MRM) analysis, given that iodothyronines are the only iodine bearing endogenous molecules. Characteristic fragments are observed at m/z 359.7 and 604.5 for rT3 but are absent in the spectrum of T3, thus differentiating the two positional isomers. The striking difference in the fragmentation patterns of these regioisomeric species is attributed to the increased acidity of the phenol moiety in rT3 compared with T3. Copyright (C) 2005 John Wiley & Sons, Ltd.

The fragmentation pathways of protonated Amiton in the gas phase : towards the structural characterisation of organophosphorus chemical warfare agents by electrospray ionisation tandem mass spectrometry

Amiton (O,O-diethyl-S-[2-(diethylamino)ethyl]phosphorothiolate), otherwise known as VG, is listed in schedule 2 of the Chemical Weapons Convention (CWC) and has a structure closely related to VX (O-ethyl-S-(2-diisopropylamino)ethylmethylphosphonothiolate). Fragmentation of protonated VG in the gas phase was performed using electrospray ionisation ion trap mass spectrometry (ESI-ITMS) and revealed several characteristic product ions. Quantum chemical calculations provide the most probable structures for these ions as well as the likely unimolecular mechanisms by which they are formed. The decomposition pathways predicted by computation are consistent with deuterium-labeling studies. The combination of experimental and theoretical data suggests that the fragmentation pathways of VG and analogous organophosphorus nerve agents, such as VX and Russian VX, are predictable and thus ESI tandem mass spectrometry is a powerful tool for the verification of unknown compounds listed in the CWC. Copyright (c) 2006 Commonwealth of Australia. Published by John Wiley & Sons, Ltd.