Liquid chromatography electron capture dissociation tandem mass spectrometry (LC-ECD-MS/MS) versus liquid chromatography collision-induced dissociation tandem mass spectrometry (LC-CID-MS/MS) for the identification of proteins

Electron capture dissociation (ECD) offers many advantages over the more traditional fragmentation techniques for the analysis of peptides and proteins, although the question remains: How suitable is ECD for incorporation within proteomic strategies for the identification of proteins? Here, we compare LC-ECD-MS/MS and LC-CID-MS/MS as techniques for the identification of proteins. Experiments were performed on a hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer. Replicate analyses of a six-protein (bovine serum albumin, apo-transferrin, lysozyme, cytochrome c, alcohol dehydrogenase, and β-galactosidase) tryptic digest were performed and the results analyzed on the basis of overall protein sequence coverage and sequence tag lengths within individual peptides. The results show that although protein coverage was lower for LC-ECD-MS/MS than for LC-CID-MS/MS, LC-ECD-MS/MS resulted in longer peptide sequence tags, providing greater confidence in protein assignment.     

Triple mass spectrometry (MS/MS/MS) with a floated collision cell in a four-sector tandem mass spectrometer

A method is described for carrying out triple mass spectrometry (MS/MS/MS) experiments with an electrically floated collision cell in the third field-free region on a tandem double-focusing mass spectrometer. The method described may use magnet calibration obtained at any accelerating voltage and is generally applicable at any value of the collision cell voltage. The utility of the method to acquire MS/MS/MS spectra of enhanced quality is demonstrated on a JEOL JMS-HX110/HX110 four-sector mass spectrometer.     

Desorption electrospray ionization (DESI) mass spectrometry and tandem mass spectrometry (MS/MS) of phospholipids and sphingolipids: Ionization, adduct formation, and fragmentation

Desorption electrospray ionization (DESI) mass spectrometry was evaluated for the characterization of glycerophospholipid standards, including glycerophosphocholine (GPCho), glycerophosphoglycerol (GPGro), glycerophosphoethanolamine (GPEtn), glycerophosphoserine (GPSer), glycerophosphoinositol (GPIns), cardiolipin (CL), and sphingolipid standards, including sulfatides (ST) and sphingomyelin (SM). Of specific interest were the effects of surface and solvent composition on signal stability and intensity, along with the ions observed in the full scan mode and the fragmentations seen upon collisional activation for each of the above classes. These experiments were performed without the addition of matrix compounds to the sample and were conducted in the free ambient environment at atmospheric pressure. The compounds GPSer, GPGro, GPIns, ST, and CL were best analyzed in the negative ion mode while PE was ionized efficiently in both positive and negative ion modes. SM and GPCho, which typically generate more abundant ions in the positive ion mode, could be analyzed in the negative ion mode by the addition of anionic reagents such as acetate to the spray solvent. Full scan DESI mass spectra and tandem (MS/MS) spectra for this representative set of physiological phospho/sphingolipids are presented. Similarities with other ionization methods in terms of fragmentation behavior were strong, although ambient ionization of untreated samples is only available with DESI. The effect of surface and solvent properties on signal intensity and stability were determined by depositing standard compounds on several different surfaces and analyzing with various proportions of methanol in the aqueous spray. Analysis was extended to complex mixtures of phospholipids and sphingolipids by examining the total lipid extract of porcine brain and by direct analysis of rat brain cryotome sections. These types of mixture analyses and molecular imaging studies are likely to represent major areas of application of DESI.     

Determination of phytochelatins by capillary zone electrophoresis with electrospray tandem mass spectrometry detection (CZE-ES MS/MS)

The coupling of capillary zone electrophoresis with electrospray mass spectrometry was optimized for the direct determination of phytochelatins (PCs) in extracts obtained from cells and plants that had been exposed to metal stress. Gluthathione and phytochelatins belonging to the different families (gamma Glu-Cys)nGly (n-PC), (gamma Glu-Cys)nSer, (gamma Glu-Cys)n beta Ala and (gamma Glu-Cys)n were separated in an uncoated capillary at pH 4 using a 5 mM ammonium acetate buffer, and detected by electrospray (ES) MS in the full scan mode (300-1100 u). The use of on-line tandem MS detection in the product ion scan mode of putative protonated molecules of PCs allowed the unambiguous confirmation of the identity of the compounds detected by ES MS. The operational conditions were optimized and the figures of merit were evaluated using n-PC2, n-PC3 and n-PC4 standards purified from a mixture obtained after the reaction of glutathione in the presence of Cd2+ and the enzyme PC-synthase. The method was applied to the characterization of bioinduced ligands in cell cultures of soybeans (Glycine max) and in rice (Oryza sativa) roots without the need for a preliminary sample cleanup by size-exclusion and/or reversed phase chromatography.     

Fragmentation of diketopiperazines from Aspergillus fumigatus by electrospray ionization tandem mass spectrometry (ESI-MS/MS)

Diketopiperazines (DKPs) corresponding to cyclic dipeptides have been reported to exhibit antimicrobial, antitumor, antimutagenic and antiviral properties. These compounds are commonly isolated from microorganisms and sponges and from a variety of tissues and body fluids. In this work, we used electrospray ionization tandem mass spectrometry (ESI-MS/MS) to investigate the fragmentation of a series of DKPs previously isolated from Aspergillus fumigatus, which exhibit the same structural core. Loss of CO directly from the protonated molecule was found to be a fragmentation process common to all the compounds analyzed. However, our results revealed a series of ions that are diagnostic for the substituents at C(4) and C(9). In order to rationalize the differences in the fragmentation pathways of substituted and nonsubstituted DKPs, the relative Gibbs energies (DeltaG) of the product ions and intermediate ions were estimated using the B3LYP/6-31 + + G(d,p) model. The data reported here can be used for the structural elucidation of DKPs from low sample amounts, as an alternative to NMR.     

Application of gas chromatography–tandem mass spectrometry (GC/MS/MS) for the analysis of deuterium enrichment of water

Incorporation of deuterium from deuterium oxide (²H₂O) into biological components is a commonly used approach in metabolic studies. Determining the dilution of deuterium in the body water (BW) pool can be used to estimate body composition. We describe three sensitive GC/MS/MS methods to measure water enrichment in BW. Samples were reacted with NaOH and U‐¹³C₃‐acetone in an autosampler vial to promote deuterium exchange with U‐¹³C₃‐acetone hydrogens. Headspace injections were made of U‐¹³C₃‐acetone‐saturated air onto a 30‐m DB‐1MS column in electron impact‐mode. Subjects ingested 30 ml ²H₂O, and plasma samples were collected. BW was determined by standard equation. Dual‐energy X‐ray absorptiometry scans were performed to calculate body mass, body volume and bone mineral content. A four‐compartmental model was used to estimate body composition (fat and fat free mass). Full‐scan experiments generated an m/z 45 peak and to a lesser extent an m/z 61 peak. Product fragment ions further monitored included 45 and 46 using selected ion monitoring (Method1), the 61 > 45 and 62 > 46 transition using multiple reaction monitoring (MRM; Method2) and the neutral loss, 62 > 45, transition (Method3). MRM methods were optimized for collision energy (CE) and collision‐induced dissociation (CID) argon gas pressure with 6 eV CE and 1.5 mTorr CID gas being optimal. Method2 was used for final determination of ²H₂O enrichment of subjects because of lower natural background. We have developed a sensitive method to determine ²H₂O enrichment in BW to enable measurement of FM and FFM. Copyright © 2015 John Wiley & Sons, Ltd.     

Ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) and UPLC/MSE analysis of RNA oligonucleotides

Fast and efficient ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) analysis of short interfering RNA oligonucleotides was used for identity confirmation of the target sequence‐related impurities. Multiple truncated oligonucleotides and metabolites were identified based on the accurate mass, and their presumed sequence was confirmed by MS/MS and MS^E (alternating low and elevated collision energy scanning modes) methods. Based on the resulting fragmentation of native and chemically modified oligonucleotides, it was found that the MS^E technique is as efficient as the traditional MS/MS method, yet MS^E is more general, faster, and capable of producing higher signal intensities of fragment ions. Fragmentation patterns of modified oligonucleotides were investigated using RNA 2′‐ribose substitutions, phosphorothioate RNA, and LNA modifications. The developed sequence confirmation method that uses the MS^E approach was applied to the analysis of in vitro hydrolyzed RNA oligonucleotide. The target RNA and metabolites, including the structural isomers, were resolved by UPLC, and their identity was confirmed by MS^E. Simultaneous RNA truncations from both termini were observed. The UPLC quadrupole time‐of‐flight (QTOF) MS/MS and MS^E methods were shown to be an effective tool for the analysis and sequence confirmation of complex oligonucleotide mixtures. Copyright © 2010 John Wiley & Sons, Ltd.     

Direct analysis of isopropylthioxanthone (ITX) in milk by high-performance liquid chromatography/tandem mass spectrometry

A fast screening method is presented for detecting isopropylthioxanthone (ITX) contamination in milk. The method is based on direct high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) analysis of milk samples. Sample preparation is limited to the addition of a deuterated ITX solution in acetonitrile that serves both as internal standard and to precipitate proteins. The method is highly accurate and sensitive. Isomeric specific analyses of 2-ITX and 4-ITX are possible at 6 microg/L levels with about 5% precision and accuracy. This approach has been used to check contamination in samples like milk, soy milk, baby milk, in their packaging material. Out of 37 milk samples analyzed, 16 were positive with concentrations ranging from 173-439 microg/L for 2-ITX and from <6 (lower than limit of quantification) to 25 microg/L for 4-ITX.     

An alternative sampling algorithm for use in liquid chromatography/tandem mass spectrometry experiments

In liquid chromatography/tandem mass spectrometry (LC/MS/MS) analyses of complex peptide mixtures, dynamic exclusion functions are used to minimize repeat selections of identical precursors for collision induced dissociation (CID). We describe a new algorithm for the dynamic exclusion of m/z values during LC/MS/MS. Full-scan based peak exclusion (Fulspec) uses a simplified model of chromatographic peak formation to detect and exclude contaminants present throughout the run or that lead to broad peaks. Therefore, instead of excluding peptides from fragment analysis according to a rigidly predefined time window, the chromatographic properties of the detected analytes are used. The algorithm was tested on two datasets derived from previously published experiments. Fulspec achieves a distribution of CID spectra with minimal tailing on the retention time axis, without resorting to rigid exclusion of m/z values. The procedure further excludes intensities with a bias towards low-quality CID spectra. This combination frees up valuable analytical capacity. The underlying intensity vs. quality analyses challenge the assumption that abundant precursors automatically give the best identifications. Further validation of the algorithm will require its incorporation by equipment manufacturers into the instrument control programs.     

Determination of acidic herbicides in fruits and vegetables using liquid chromatography tandem mass spectrometry (LC-MS/MS)

The use of quick, easy, cheap, effective, rugged and safe method followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) was found to be the best combination for multiresidue determination of eight acidic herbicides in fruits and vegetables in terms of high recovery, short time of analysis, low cost and safety. Recent few articles were published for determination of different classes of acidic herbicides in single multiresidue method. In the present study, mass spectrophotometric conditions were individually optimised for eight acidic herbicides, namely 2,4-dichlorophenoxyacetic acid, bentazone, bromoxynil, fluazifop, fluroxypyr, imazethapyr, ioxynil and triclopyr to achieve maximum sensitivity and selectivity in multiple reaction monitoring (MRM) mode allowing simultaneous identification and quantification in a single run. Identity confirmation and quantitation were attained by using negative electrospray ionisation LC-MS/MS (ESI?) in MRM mode. Due to LC-MS/MS signal suppression, determination of pesticide residues was based on matrix-matched standard calculations. Most of the evaluated compounds showed a recovery ranging from 81% to 113% with relative standard deviations less than 16 % indicating acceptable precision. The precision and accuracy of the method were determined from recovery experiments on six replicates of spiked blank strawberry and green beans samples at 0.01, 0.05 and 0.1 mg/kg. The developed assay was linear over concentration range of 0.01–0.5 µg/mL, with correlation coefficient greater than 0.99 at the limit of quantitation 0.01 µg/mL. The proposed assay was successfully applied for the analysis of the studied acidic herbicides residues in two proficiency test samples. This wide scope assay protocol is applicable for monitoring acidic herbicides residues in fruits and vegetables by national regulatory authorities and accredited labs in order to help ensuring the safety of such widely used food products.     

Quantitation of fluoroquinolones in honey using tandem mass spectrometry (LC-MS/MS): nested validation with two mass spectrometers

A number of drugs in the quinolone and fluoroquinolone families, approved for veterinary treatment of food animals by various countries, may be used to treat bee diseases and thereby contaminate honey. An LC-MS/MS method has been developed for the quantification of the quinolones: flumequine, nalidixic acid, oxolinic acid, and pipemidic acid; and the fluoroquinolones ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, norfloxacin, ofloxacin, orbifloxacin, marbofloxacin, sarafloxacin, and sparfloxacin. A method-matched calibration curve is used with several internal standards, i.e., ciprofloxacin-d8, Iomefloxacin, and cinoxacin, to correct for the various types of honey matrices: white, light, medium, and dark colors. Enoxacin is added as an external recovery standard. The LOD values range from 0.05 microg/kg (ofloxacin) to 0.4 microg/kg (flumequine). The compounds are verified by LC-MS/MS retention times and ion ratios. Method uncertainty was determined using two separate analytical systems. The method has successfully measured the presence of norfloxacin in several samples of honey imported into Canada.     

High‐speed MALDI MS/MS imaging mass spectrometry using continuous raster sampling

A matrix‐assisted laser desorption/ionization time of flight/time of flight tandem mass spectrometer (MALDI TOF/TOF) has been used for high‐speed precursor/fragment ion transition image acquisition. High‐throughput analysis is facilitated by an Nd:YLF solid state laser capable of pulse repetition rates up to 5 kHz, a high digitizer acquisition rate (up to 50 pixels/s), and continuous laser raster sampling. MS/MS experiments are enabled through the use of a precision timed ion selector, second source acceleration, and a dedicated collision cell. Continuous raster sampling is shown here to facilitate rapid MS/MS ion image acquisition from thin tissue sections for the drug rifampicin and for a common kidney lipid, SM4s(d18:1/24:1). The ability to confirm the structural identity of an analyte as part of the MS/MS imaging experiment is an essential part of the analysis. Additionally, the increase in sensitivity and specificity afforded by an MS/MS approach is highly advantageous, especially when interrogating complex chemical environments such as those in biological tissues. Herein, we report continuous laser raster sampling TOF/TOF imaging methodologies which demonstrate 8 to 14‐fold increases in throughput compared with existing MS/MS instrumentation, an important advantage when imaging large areas on tissues. Copyright © 2015 John Wiley & Sons, Ltd.     

Structural characterization and isomer differentiation of chalcones by electrospray ionization tandem mass spectrometry

A series of chalcones were characterized by electrospray ionization tandem mass spectrometry (MS(n)). Several ionization modes were evaluated, including protonation, deprotonation and metal complexation, with metal complexation being the most efficient. Collision-activated dissociation (CAD) was used to characterize the structures, and losses commonly observed include H(2), H(2)O, CO and CO(2), in addition to methyl radicals for the methoxy-containing chalcones. CAD of the metal complexes, especially [Co(II) (chalcone-H) 2,2'-bipyridine](+), allowed the most effective differentiation of the isomeric chalcones with several diagnostic fragment ions appearing upon activation of the metal complexes. MS(n) experiments were performed to support identification of some fragment ions and to verify the proposed fragmentation pathways. In several cases, MS(n) indicated that specific neutral losses occurred by stepwise pathways, such as the neutral loss of 44 u as CH3* and HCO*, or CH(4) and CO, in addition to CO(2).     

Studies of phthalic acid and related compounds by positive- and negative-ion chemical ionization tandem mass spectrometry (± CI/MS/MS)

The positive- and negative-ion chemical ionization (± CI) behavior of phthalic acid and four related compounds (phthalic anhydride, phthalide, biphthalyl, and anthraquinone-2-carboxyIic acid) are characterized by tandem mass spectrometry with low-energy collisionally activated dissociation. Principal fragmentation pathways have been established, and molecular and fragment ions arising from these compounds have been differentiated from adduct and dimer ions, as well as from ions due to impurities. The ubiquitous nature of these compounds in analytical samples makes it important to identify the corresponding ions, and thus to be able to exclude them from consideration in the identification of other sample components.     

Measurement of bile acid CoA esters by high-performance liquid chromatography-electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS)

The novel and rapid assay presented here combines high-performance liquid chromatography and electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS) to directly measure and quantify the CoA esters of 3alpha,7alpha,12alpha-trihydroxy- and 3alpha,7alpha-dihydroxy-5beta-cholestan-26-oic acid (THCA and DHCA). The latter are converted inside peroxisomes to the primary bile acids, cholic and chenodeoxycholic acids, respectively. Prior to MS/MS, esters were separated by reversed-phase HPLC on a C(18) column using an isocratic mobile phase (acetonitrile/water/2-propanol) and subsequently detected by multiple reaction monitoring. For quantification, the CoA ester of deuterium-labelled 3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oic acid (d(4)-CA) was used as internal standard. To complete an assay took less than 8 min.To verify the validity of the assay, the effect of peroxisomal proteins on the efficacy of extraction of the CoA esters was tested. To this end, variable amounts of the CoA esters were spiked with a fixed amount of either intact peroxisomes or peroxisomal matrix proteins and then extracted using a solid-phase extraction system. The CoA esters could be reproducibly recovered in the range of 0.1-4 micromol l(-1) (linear correlation coefficient R(2) > 0.99), with a detection limit of 0.1 micromol l(-1).In summary, electrospray ionization tandem mass spectrometry combined with HPLC as described here proved to be a rapid and versatile technique for the determination of bile acid CoA esters in a mixture with peroxisomal proteins. This suggests this technique to become a valuable tool in studies dealing with the multi-step biosynthesis of bile acids and its disturbances in disorders like the Zellweger syndrome.     

Membrane preconcentration-capillary electrophoresis tandem mass spectrometry (mPC-CE-MS/MS) in the sequence analysis of biologically derived peptides

We demonstrate the use of membrane preconcentration capillary electrophoresis tandem mass spectrometry (mPC-CE-MS/MS) for sequencing peptides at the sub-100 femtomole level. In particular by loading the mPC-CE cartridge off-line with a pressurized bomb apparatus, 100 mul solutions can be loaded in <5 min. Furthermore, mPC-CE-MS in conjunction with on-line transient isotachophoresis carried out in 25 mum i.d. capillaries results in enhanced resolution and theoretical plate values as compared to convention 50-75 mum i.d. capillaries. We show that this is a powerful new approach in the sequencing of biologically derived compounds from complex mixtures such as MHC class I peptides.