Development and validation of a method for the quantification of milk proteins in food products based on liquid chromatography with mass spectrometric detection

The protection of allergic consumers is crucial to the food industry. Therefore, accurate methods for the detection of food allergens are required. Targeted detection of selected molecules by MS combines high selectivity with accurate quantification. A confirmatory method based on LC/selected reaction monitoring (SRM)-MS/MS was established and validated for the quantification of milk traces in food. Tryptic peptides of the major milk proteins beta-lactoglobulin, beta-casein, alphaS2-casein, and K-casein were selected as quantitative markers. Precise quantification was achieved using internal standard peptides containing isotopically labeled amino acids. For each peptide, qualifier and quantifier fragments were selected according to Commission Decision 2002/657/EC. A simple sample preparation method was established without immunoaffinity or SPE enrichment steps for food matrixes containing different amounts of protein, such as baby food, breakfast cereals, infant formula, and cereals. Intermediate reproducibility, repeatability, accuracy, and measurement uncertainty were determined for each matrix. LOD values of 0.2-0.5 mg/kg, e.g., for beta-lactoglobulin, were comparable to those obtained with ELISA kits. An LOQ of approximately 5 mg/kg, expressed as mass fraction skim milk powder, was validated in protein-rich infant cereals. The obtained validation data show that the described LC/SRM-MS/MS approach can serve as a confirmatory method for the determination of milk traces in selected food matrixes.     

Identification of allergenic milk proteins markers in fined white wines by capillary liquid chromatography–electrospray ionization-tandem mass spectrometry

A method based on capillary liquid chromatography combined with electrospray ionization-tandem mass spectrometry (CapLC–ESI-MS–MS) for the detection and identification of casein deriving peptides in fined white wine is described. This is the first step towards the development of a liquid chromatography mass spectrometric method for the detection/identification of markers of potentially allergenic milk proteins used as wine fining agents. The method demonstrated to be capable of detecting some peptides arising from α and β casein (with the relative aminoacidic sequences elucidated) in extracts of white wine fined with casein at 100 and 1000 μg/mL. This MS based approach appears to be a useful tool for screening purposes as well as a confirmatory tool for the unequivocal identification of caseins in ELISA positive samples.     

Precise and global identification of phospholipid molecular species by an Orbitrap mass spectrometer and automated search engine Lipid Search

In the present research, we have established a new lipidomics approach for the comprehensive and precise identification of molecular species in a crude lipid mixture using a LTQ Orbitrap mass spectrometer (MS) and reverse-phase liquid chromatography (RPLC) combination with our newly developed lipid search engine “Lipid Search”. LTQ Orbitrap provides high mass accuracy MS spectra by Fourier-transform (FT) mass spectrometer mode and can perform rapid MSⁿ by ion trap (IT) mass spectrometer mode. In this study, the negative ion mode was selected to detect fragment ions from phospholipids, such as fatty acid anions, by MS2 or MS3. We selected the specific detection approach by neutral loss survey-dependent MS3, for the identification of molecular species of phosphatidylcholine, sphingomyelin and phosphatidylserine. Identification of molecular species was performed by using both the high mass accuracy of the mass spectrometric data obtained from FT mode and structural data obtained from fragments in IT mode. Some alkylacyl and alkenylacyl species have the same m/z value as molecular-related ions and fragment ions, thus, direct acid hydrolysis analysis was performed to identify alkylacyl and alkenylacyl species, and then the RPLC–LTQ Orbitrap method was applied. As a result, 290 species from mouse liver and 248 species from mouse brain were identified within six different classes of phospholipid, only those in manually detected and confirmed. Most of all manually detected mass peaks were also automatically detected by “Lipid Search”. Adding to differences in molecular species in different classes of phospholipids, many characteristic differences in molecular species were detected in mouse liver and brain. More variable number of saturated and monounsaturated fatty acid-containing molecular species were detected in mouse brain than liver.     

Quantitation of small molecules using high‐resolution accurate mass spectrometers – a different approach for analysis of biological samples

The quantitative capabilities of a linear ion trap high‐resolution mass spectrometer (LTQ‐Orbitrap™) were investigated using full scan mode bracketing the m/z range of the ions of interest and utilizing a mass resolution (mass/FWHM) of 15000. Extracted ion chromatograms using a mass window of ±5–10 mmu centering on the theoretical m/z of each analyte were generated and used for quantitation. The quantitative performance of the LTQ‐Orbitrap™ was compared with that of a triple quadrupole (API 4000) operating using selected reaction monitoring (SRM) detection. Comparable assay precision, accuracy, linearity and sensitivity were observed for both approaches. The concentrations of actual study samples from 15 Merck drug candidates reported by the two methods were statistically equivalent. Unlike SRM being a tandem mass spectrometric (MS/MS)‐based detection method, a high resolution mass spectrometer operated in full scan does not need MS/MS optimization. This approach not only provides quantitative results for compounds of interest, but also will afford data on other analytes present in the sample. An example of the identification of a major circulating metabolite for a preclinical development study is demonstrated. Copyright © 2009 John Wiley & Sons, Ltd.     

Bottom-up proteomics of envelope proteins extracted from spinach chloroplast via high organic content CE-MS

A high organic content CE-MS/MS (HOCE-MS/MS) method was developed for the proteomic analysis of envelope proteins extracted from spinach leaves. Separation was performed in a 1-m long hydroxypropyl cellulose coated capillary, using 8% (v/v) formic acid in 70% (v/v) methanol and 22% water as the BGE. A flow-through microvial interface was used to couple the CE system with an Orbitrap Fusion Lumos mass spectrometer, and field-amplified sample stacking was used to improve the concentration sensitivity. Using this optimized method, 3579 peptides and 1141 proteins were identified using the Proteome Discoverer software with a 1% false discovery rate at the protein level. Relative to conventional aqueous CE, HOCE-MS did a better job of discovering hydrophobic peptides and provided more peptide and protein identifications. Relative to nano-LC-MS, it achieved comparable peptide and protein identification performance and detected peptides not identified by LC-MS: of the full set of peptides identified using the two techniques, 19% were identified only using HOCE-MS. It also outperformed nano-LC-MS with respect to the detection of low molecular weight peptides.     

超高效液相色谱-线性离子阱/静电场轨道阱高分辨质谱直接分析葡萄酒中38种多酚类化合物

建立了超高效液相色谱-线性离子阱/静电场轨道阱高分辨质谱检测葡萄酒中38种多酚化合物的检测方法。样品过聚醚砜(PES)滤膜后直接上样分析,Hypersil Gold C18色谱柱分离,以乙腈(含0.1%甲酸)和0.1%甲酸水作为流动相梯度洗脱。在m/z 50~1000范围内进行一级质谱全扫描。以准分子离子峰的精确质量数和提取的色谱图峰面积进行筛查分析和定量,以保留时间和数据依赖扫描(data-dependent scan)模式获得的子离子质谱图进行定性确证。38种多酚化合物的质量偏差不大于5×10^-6(5 ppm),浓度与特征离子峰面积的线性关系良好(浓度线性范围为两个数量级),相关系数(R²)大于0.99,方法检出限为0.002~0.50 mg/kg。3个添加水平的回收率范围为90%~102%,相对标准偏差为0.51%~2.56%。应用该方法检测了葡萄酒中38种多酚化合物的含量,该方法准确、可靠。     

'Information-Based-Acquisition' (IBA) technique with an ion-trap/time-of-flight mass spectrometer for high-throughput and reliable protein profiling

Highly complex protein mixtures can be analyzed after proteolysis using liquid chromatography/mass spectrometry (LC/MS). In an LC/MS run, intense peptide ions originating from high-abundance proteins are preferentially analyzed using tandem mass spectrometry (MS(2)), so obtaining the MS(2) spectra of peptide ions from low-abundance proteins is difficult even if such ions are detected. Furthermore, the MS(2) spectra may produce insufficient information to identify the peptides or proteins. To solve these problems, we have developed a real-time optimization technique for MS(2), called the Information-Based-Acquisition (IBA) system. In a preliminary LC/MS run, a few of the most intense ions detected in every MS spectrum are selected as precursors for MS(2) and their masses, charge states and retention times are automatically registered in an internal database. In the next run, a sample similar to that used in the first run is analyzed using database searching. Then, the ions registered in the database are excluded from the precursor ion selection to avoid duplicate MS(2) analyses. Furthermore, real-time de novo sequencing is performed just after obtaining the MS(2) spectrum, and an MS(3) spectrum is obtained for accurate peptide identification when the number of interpreted amino acids in the MS(2) spectrum is less than five. We applied the IBA system to a yeast cell lysate which is a typical crude sample, using a nanoLC/ion-trap time-of flight (IT/TOF) mass spectrometer, repeating the same LC/MS run five times. The obtained MS(2) and MS(3) spectra were analyzed by applying the Mascot (Matrix Science, Boston, MA, USA) search engine to identify proteins from the sequence database. The total number of identified proteins in five LC/MS runs was three times higher than that in the first run and the ion scores for peptide identification also significantly increased, by about 70%, when the MS(3) spectra were used, combined with the MS(2) spectra, before being subjected to Mascot analysis.     

MALDI produced ions inspected with a linear ion trap-orbitrap hybrid mass analyzer

A MALDI source is interfaced to a modified LTQ Orbitrap XL instrument. This work gives insight into the MALDI source design and shows results obtained with the MALDI source coupled to an accurate mass, high-resolution hybrid mass spectrometer. MALDI-produced ions and fragment ions thereof produced in the mass spectrometer may be analyzed and detected by the Orbitrap analyzer at a maximum mass resolution of 100,000 (FWHM) at m/z 400 with high mass accuracy. An accuracy of ≤2 ppm is achieved by internal mass calibration using lock mass functionality; using external mass calibration, an accuracy of ≤3 ppm is routinely obtained. External mass calibration of the hybrid mass spectrometer is performed using a standard calibration mixture of different peptides and matrix components. The instrumental capabilities are demonstrated for analytical methodologies such as Protein ID using Peptide Mass Fingerprint (PMF) and MS/MS analyses of small molecule samples. Stability of mass accuracy and signal-to-noise ratio for low samples loads (on plates) are demonstrated as well as the experimental dynamic range using α-cyano-4-hydroxy cinnamic acid (CHCA) matrix.     

New and automated MS<Superscript>n</Superscript> approaches for top-down identification of modified proteins

An automated top-down approach including data-dependent MS(3) experiment for protein identification/characterization is described. A mixture of wild-type yeast proteins has been separated on-line using reverse-phase liquid chromatography and introduced into a hybrid linear ion trap (LTQ) Fourier transform ion cylclotron resonance (FTICR) mass spectrometer, where the most abundant molecular ions were automatically isolated and fragmented. The MS(2) spectra were interpreted by an automated algorithm and the resulting fragment mass values were uploaded to the ProSight PTM search engine to identify three yeast proteins, two of which were found to be modified. Subsequent MS(3) analyses pinpointed the location of these modifications. In addition, data-dependent MS(3) experiments were performed on standard proteins and wild-type yeast proteins using the stand alone linear trap mass spectrometer. Initially, the most abundant molecular ions underwent collisionally activated dissociation, followed by data-dependent dissociation of only those MS(2) fragment ions for which a charge state could be automatically determined. The resulting spectra were processed to identify amino acid sequence tags in a robust fashion. New hybrid search modes utilized the MS(3) sequence tag and the absolute mass values of the MS(2) fragment ions to collectively provide unambiguous identification of the standard and wild-type yeast proteins from custom databases harboring a large number of post-translational modifications populated in a combinatorial fashion.     

Multiresidue pesticide analysis in ginseng and spinach by nontargeted and targeted screening procedures

Five different mass spectrometers interfaced to GC or LC were evaluated for their application to targeted and nontargeted screening of pesticides in two foods, spinach and ginseng. The five MS systems were capillary GC/MS/MS, GC-high resolution time-of-flight (GC/HR-TOF)-MS, TOF-MS interfaced with a comprehensive multidimensional GC (GCxGC/TOF-MS), an MS/MS ion trap hybrid mass (qTrap) system interfaced with an ultra-performance liquid chromatograph (UPLC-qTrap), and UPLC interfaced to an orbital trap high resolution mass spectrometer (UPLC/Orbitrap HR-MS). Each MS system was tested with spinach and ginseng extracts prepared through a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) procedure. Each matrix was fortified at 10 and 50 ng/g for spinach or 25 and 100 ng/g for ginseng with subsets of 486 pesticides, isomers, and metabolites representing most pesticide classes. HR-TOF-MS was effective in a targeted search for characteristic accurate mass ions and identified 97% of 170 pesticides in ginseng at 25 ng/g. A targeted screen of either ginseng or spinach found 94-95% of pesticides fortified for analysis at 10 ng/g with GC/MS/MS or LC/MS/MS using multiple reaction monitoring (MRM) procedures. Orbitrap-MS successfully found 89% of 177 fortified pesticides in spinach at 25 ng/g using a targeted search of accurate mass pseudomolecular ions in the positive electrospray ionization mode. A comprehensive GCxGC/TOF-MS system provided separation and identification of 342 pesticides and metabolites in a single 32 min acquisition with standards. Only 67 or 81% of the pesticides were identified in ginseng and spinach matrixes at 25 ng/g or 10 ng/g, respectively. MS/MS or qTrap-MS operated in the MRM mode produced the lowest false-negative rates, at 10 ng/g. Improvements to instrumentation, methods, and software are needed for efficient use of nontargeted screens in parallel with triple quadrupole MS.     

Comparison of different signal thresholds on data dependent sampling in orbitrap and LTQ mass spectrometry for the identification of peptides and proteins in complex mixtures

We evaluate the effect of ion-abundance threshold settings for data-dependent acquisition on a hybrid LTQ-Orbitrap mass spectrometer, analyzing features such as the total number of spectra collected, the signal to noise ratio of the full MS scans, the spectral quality of the tandem mass spectra acquired, and the number of peptides and proteins identified from a complex mixture. We find that increasing the threshold for data-dependent acquisition generally decreases the quantity but increases the quality of the spectra acquired. This is especially true when the threshold setting is set above the noise level of the full MS scan. We compare two distinct experimental configurations: one where full MS scans are acquired in the Orbitrap analyzer while tandem MS scans are acquired in the LTQ analyzer, and one where both full MS and tandem MS scans are acquired in the LTQ analyzer. We examine the number of spectra, peptides, and proteins identified under various threshold conditions, and we find that the optimal threshold setting is at or below the respective noise level of the instrument regardless of whether the full MS scan is performed in the Orbitrap or in the LTQ analyzer. When comparing the high-throughput identification performance of the two analyzers, we conclude that, used at optimal threshold levels, the LTQ and the Orbitrap identify similar numbers of peptides and proteins. The higher scan speed of the LTQ, which results in more spectra being collected, is roughly compensated by the higher mass accuracy of the Orbitrap, which results in improved database searching and peptide validation software performance.     

Resolving the composition of protein complexes using a MALDI LTQ orbitrap

Current biological studies have been advanced by the continuous development of robust, accurate, and sensitive mass spectrometric technologies. The MALDI LTQ Orbitrap is a new addition to the Orbitrap configurations, known for their high resolving power and accuracy. This configuration provides features inherent to the MALDI source, such as reduced spectra complexity, forgiveness to contaminants, and sample retention for follow-up analyses with targeted or hypothesis-driven questions. Here we investigate its performance for characterizing the composition of isolated protein complexes. To facilitate the assessment, we selected two well characterized complexes from Saccharomyces cerevisiae, Apl1 and Nup84. Manual and automatic MS and MS/MS analyses readily resolved their compositions, with increased confidence of protein identification compared with our previous reports using MALDI QqTOF and MALDI IT. CID fragmentation of singly-charged peptides provided sufficient information for conclusive identification of the isolated proteins. We then assessed the resolution, accuracy, and sensitivity provided by this instrument in the context of analyzing the isolated protein assemblies. Our analysis of complex mixtures of singly-charged ions up to m/z 4000 showed that (1) the resolving power, inversely proportional to the square root of m/z, had over four orders of magnitude dynamic range; (2) internal calibration led to improved accuracy, with an average absolute mass error of 0. 5 ppm and a distribution centered at 0 ppm; and (3) subfemtomole sensitivity was achieved using both CHCA and DHB matrices. Additionally, our analyses of a synthetic phosphorylated peptide in mixtures showed subfemtomole level of detection using neutral loss scanning.     

"De novo" peptide sequencing by MALDI-quadrupole-ion trap mass spectrometry: a preliminary study

Collision-induced dissociation of singly charged peptide ions produced by resonant excitation in a matrix-assisted laser desorption/ionization (MALDI) ion trap mass spectrometer yields relatively low complexity MS/MS spectra that exhibit highly preferential fragmentation, typically occurring adjacent to aspartyl, glutamyl, and prolyl residues. Although these spectra have proven to be of considerable utility for database-driven protein identification, they have generally been considered to contain insufficient information to be useful for extensive de novo sequencing. Here, we report a procedure for de novo sequencing of peptides that uses MS/MS data generated by an in-house assembled MALDI-quadrupole-ion trap mass spectrometer (Krutchinsky, Kalkum, and Chait Anal. Chem. 2001, 73, 5066-5077). Peptide sequences of up 14 amino acid residues in length have been deduced from digests of proteins separated by SDS-PAGE. Key to the success of the current procedure is an ability to obtain MS/MS spectra with high signal-to-noise ratios and to efficiently detect relatively low abundance fragment ions that result from the less favorable fragmentation pathways. The high signal-to-noise ratio yields sufficiently accurate mass differences to allow unambiguous amino acid sequence assignments (with a few exceptions), and the efficient detection of low abundance fragment ions allows continuous reads through moderately long stretches of sequence. Finally, we show how the aforementioned preferential cleavage property of singly charged ions can be used to facilitate the de novo sequencing process.     

Characterizing protein glycosylation sites through higher‐energy C‐trap dissociation

Assigning glycosylation sites of glycoproteins and their microheterogeneity is still a very challenging analytical task despite the rapid advancements in mass spectrometry. It is shown here that glycopeptide ions can be fragmented efficiently using the higher‐energy C‐trap dissociation (HCD) feature of a linear ion trap orbitrap hybrid mass spectrometer (LTQ Orbitrap). An attractive aspect of this dissociation option is the generation of distinct Y1 ions (peptide+GlcNAc), thus allowing unequivocal assignment of N‐glycosylation sites of glycoproteins. The combination of the very informative collision‐induced dissociation spectra acquired in the linear ion trap with the distinct features of HCD offers very useful information aiding in the characterization of the glycosylation sites of glycoproteins. The HCD activation energy needed to obtain optimum Y1 ions was studied in terms of glycan structure and charge state, and size and structure of the peptide backbone. The latter appeared to be primarily dictating the needed HCD energy. The distinct Y1 ion formation in HCD facilitated an easy assignment of such an ion and its subsequent isolation and dissociation through multiple‐stage tandem mass spectrometry. The resulting MS³ spectrum of the Y1 ion facilitates database searching and de novo sequencing thus prompting the subsequent identification of the peptide backbone and associated glycosylation sites. Moreover, fragment ions formed by HCD are detected in the Orbitrap, thus overcoming the 1/3 cut‐off limitation that is commonly associated with ion trap mass spectrometers. As a result, in addition to the Y1 ion, the common glycan oxonium ions are also detected. The high mass accuracy offered by the LTQ Orbitrap mass spectrometer is also an attractive feature that allows a confident assignment of protein glycosylation sites and the microheterogeneity of such sites. Copyright © 2010 John Wiley & Sons, Ltd.     

The nature of collision-induced dissociation processes of doubly protonated peptides: comparative study for the future use of matrix-assisted laser desorption/ionization on a hybrid quadrupole time-of-flight mass spectrometer in proteomics.

Comparative MS/MS studies of singly and doubly charged electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) precursor peptide ions are described. The spectra from these experiments have been evaluated with particular emphasis on the data quality for subsequent data processing and protein/amino acid sequence identification. It is shown that, once peptide ions are formed by ESI or MALDI, their charge state, as well as the collision energy, is the main parameter determining the quality of collision-induced dissociation (CID) MS/MS fragmentation spectra of a given peptide. CID-MS/MS spectra of singly charged peptides obtained on a hybrid quadrupole orthogonal time-of-flight mass spectrometer resemble very closely spectra obtained by matrix-assisted laser desorption/ionization post-source decay time-of-flight mass spectrometry (MALDI-PSD-TOFMS). On the other hand, comparison of CID-MS/MS spectra of either singly or doubly charged ion species shows no dependence on whether ions have been formed by ESI or MALDI. This observation confirms that, at the time of precursor ion selection, further mass analysis is effectively decoupled from the desorption/ionization event. Since MALDI ions are predominantly formed as singly charged species and ESI ions as doubly charged, the associated difference in the spectral quality of MS/MS spectra as described here imposes direct consequences on data processing, database searching using ion fragmentation data, and de novo sequencing when ionization techniques are changed.     

Charge state separation for protein applications using a quadrupole time-of-flight mass spectrometer

A novel method for separating ions according to their charge state using a quadrupole time-of-flight mass spectrometer is presented. The benefits of charge state separation are particularly apparent in protein identification applications at low femtomole concentration levels, where in conventional TOF MS spectra peptide ions are often lost in a sea of chemical noise. When doubly and triply charged tryptic peptide ions need to be filtered from singly charged background ions, the latter are suppressed by two to three orders of magnitude, while from 10-50% of multiply charged ions remain. The suppression of chemical noise reduces the need for chromatography and can make this experimental approach the electrospray equivalent of conventional MALDI peptide maps. If unambiguous identification cannot be achieved, MS/MS experiments are performed on the precursor ions identified through charge separation, while the previously described Q2-trapping duty cycle enhancement is tuned for approximately 1.4 of the precursor m/z to enhance intensities of ions with m/z values above that of the precursor. The resulting product ion spectra contain few fragments of impurities and provide quick and unambiguous identification through database search. The multiple charge separation technique requires minimal tuning and may become a useful tool for analysis of complex mixtures.     

LC/MS and LC/MS/MS based protocol for identification of dyes in historic textiles

Identification of dyes in historic textiles was until recently only based on reversed phase liquid chromatography and diode-array detection (RPLC–DAD). Although in the last years mass spectrometry (MS) is increasingly used as a detection system for liquid chromatography, most applications in the field are directed to identification of the molecular ions or in studies dedicated to degradation products which may be used as markers in RPLC–DAD. In the present work, an analytical protocol for the identification of dyes using RPLC/ESI/MS is presented. Atmospheric pressure electrospray ionization (ESI) was applied, in the negative ion monitoring mode. Both single stage and tandem MS (MS/MS) approaches were considered. An ion trap was used as mass analyzer. Experiments are based on the characterization of standards (natural dyes and/or dyed fibers) with the mass spectrometer sequentially working in the following modes: single MS/full scan, followed by plotting chromatograms through ion extraction (IEC) according to mass/charge ratios corresponding to molecular ions; single MS/selected ion monitoring (SIM) mode; tandem MS/single reaction monitoring (SRM) mode; tandem MS/multiple reactions monitoring (MRM) or product ion scanning modes. A faster chromatographic separation could be applied as MS detection readily balanced the selectivity of the analytical process. In a case study, 11 dyes from 3 biological sources were detected in a 0.5 mg historic sample.     

Matrix-assisted laser desorption/ionization directed nano-electrospray ionization tandem mass spectrometric analysis for protein identification

In those cases where the information obtained by peptide mass fingerprinting or matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) is not sufficient for unambiguous protein identification, nano-electrospray ionization (nano-ESI) and/or electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis must be performed. The sensitivity of nano-ESI/MS, however, is lower than that of MALDI-MS, especially at very low analyte concentrations and/or in the presence of contaminants, such as salt and detergents. Moreover, to perform ESI-MS/MS, the peptide masses of the precursor ions must be known. The approach described in this paper, MALDI-directed nano-ESI-MS/MS, makes use of information obtained from the more sensitive MALDI-MS experiments in order to direct subsequent nano-ESI-MS/MS experiments. Peptide molecular ions found in the MALDI-MS analysis are then selected, as their (+2) precursor ions, for nano-ESI-MS/MS sequencing, even though these ions cannot be detected in the ESI-MS spectra. This method, originally proposed by Tempst et al. (Anal. Chem. 2000, 72: 777-790), has been extended to provide better sensitivity and shorter analysis times; also, a comparison with liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been performed. These experiments, performed using quadrupole time-of-flight instruments equipped with commercially available nano-ESI sources, have allowed the unambiguous identification of in-gel digested proteins at levels below their ESI-MS detection limits, even in the presence of salts and detergents.     

Selective identification and quantitative analysis of methionine containing peptides by charge derivatization and tandem mass spectrometry

To enable the development of a tandem mass spectrometry (MS/MS) based methodology for selective protein identification and differential quantitative analysis, a novel derivatization strategy is proposed, based on the formation of a "fixed-charge" sulfonium ion on the side-chain of a methionine amino acid residue contained within a protein or peptide of interest. The gas-phase fragmentation behavior of these side chain fixed charge sulfonium ion containing peptides is observed to result in exclusive loss of the derivatized side chain and the formation of a single characteristic product ion, independently of charge state or amino acid composition. Thus, fixed charge containing peptide ions may be selectively identified from complex mixtures, for example, by selective neutral loss scan mode MS/MS methods. Further structural interrogation of identified peptide ions may be achieved by subjecting the characteristic MS/MS product ion to multistage MS/MS (MS3) in a quadrupole ion trap mass spectrometer, or by energy resolved "pseudo" MS3 in a triple quadrupole mass spectrometer. The general principles underlying this fixed charge derivatization approach are demonstrated here by MS/MS, MS3 and "pseudo" MS3 analysis of side chain fixed-charge sulfonium ion derivatives of peptides containing methionine formed by reaction with phenacylbromide. Incorporation of "light" and "heavy" isotopically encoded labels into the fixed-charge derivatives facilitates the application of this method to the quantitative analysis of differential protein expression, via measurement of the relative abundances of the neutral loss product ions generated by dissociation of the light and heavy labeled peptide ions. This approach, termed "selective extraction of labeled entities by charge derivatization and tandem mass spectrometry" (SELECT), thereby offers the potential for significantly improved sensitivity and selectivity for the identification and quantitative analysis of peptides or proteins containing selected structural features, without requirement for extensive fractionation or otherwise enrichment from a complex mixture prior to analysis.