Evaluation of multiple alternative instrument platforms for targeted and non‐targeted dioxin and furan analysis

The use of gas chromatography coupled to high‐resolution magnetic sector mass spectrometers (GC‐HRMS) is well established for dioxin and furan analysis. However, the use of gas chromatography coupled to triple quadrupole (MS/MS) and time of flight (TOF) mass spectrometers with atmospheric pressure ionization (API) and traditional electron ionization (EI) for dioxin and furan analysis is emerging as a viable alternative to GC‐HRMS screening. These instruments offer greater versatility in the lab for a wider range of compound identification and quantification as well as improved ease of operation. The instruments utilized in this study included 2 API‐MS/MS, 1 traditional EI‐MS/MS, an API‐quadrupole time of flight mass spectrometer (API‐QTOF), and a EI‐high‐resolution TOF (EI‐HRTOF). This study compared these 5 instruments to a GC‐HRMS using method detection limit (MDLs) samples for dioxin and furan analysis. Each instrument demonstrated acceptable MDL values for the 17 chlorinated dioxin and furans studied. The API‐MS/MS instruments provide the greatest overall improvement in MDL value over the GC‐HRMS with a 1.5 to 2‐fold improvement. The API‐QTOF and EI‐TOF demonstrate slight increases in MDL value as compared with the GC‐HRMS with a 1.5‐fold increase. The 5 instruments studied all demonstrate acceptable MDL values with no MDL for a single congener greater than 5 times that for the GC‐HRMS. All 5 instruments offer a viable alternative to GC‐HRMS for the analysis of dioxins and furans and should be considered when developing new validated methodologies.     

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.     

Quantitative mapping of glycoprotein micro‐heterogeneity and macro‐heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides

Mass spectrometry (MS) is used to quantify the relative distribution of glycans attached to particular protein glycosylation sites (micro‐heterogeneity) and evaluate the molar site occupancy (macro‐heterogeneity) in glycoproteomics. However, the accuracy of MS for such quantitative measurements remains to be clarified. As a key step towards this goal, a panel of related tryptic peptides with and without complex, biantennary, disialylated N‐glycans was chemically synthesised by solid‐phase peptide synthesis. Peptides mimicking those resulting from enzymatic deglycosylation using PNGase F/A and endo D/F/H were synthetically produced, carrying aspartic acid and N‐acetylglucosamine‐linked asparagine residues, respectively, at the glycosylation site. The MS ionisation/detection strengths of these pure, well‐defined and quantified compounds were investigated using various MS ionisation techniques and mass analysers (ESI‐IT, ESI‐Q‐TOF, MALDI‐TOF, ESI/MALDI‐FT‐ICR‐MS). Depending on the ion source/mass analyser, glycopeptides carrying complex‐type N‐glycans exhibited clearly lower signal strengths (10–50% of an unglycosylated peptide) when equimolar amounts were analysed. Less ionisation/detection bias was observed when the glycopeptides were analysed by nano‐ESI and medium‐pressure MALDI. The position of the glycosylation site within the tryptic peptides also influenced the signal response, in particular if detected as singly or doubly charged signals. This is the first study to systematically and quantitatively address and determine MS glycopeptide ionisation/detection strengths to evaluate glycoprotein micro‐heterogeneity and macro‐heterogeneity by label‐free approaches. These data form a much needed knowledge base for accurate quantitative glycoproteomics. Copyright © 2013 John Wiley & Sons, Ltd.     

Modern MALDI time‐of‐flight mass spectrometry

This paper focuses on development of time‐of‐flight (TOF) mass spectrometry in response to the invention of matrix‐assisted laser desorption/ionization (MALDI). Before this breakthrough ionization technique for nonvolatile molecules, TOF was generally considered as a useful tool for exotic studies of ion properties but was not widely applied to analytical problems. Improved TOF instruments and software that allow the full potential power of MALDI to be applied to difficult biological applications are described. A theoretical approach to the design and optimization of MALDI‐TOF instruments for particular applications is presented. Experimental data are provided that are in excellent agreement with theoretical predictions of resolving power and mass accuracy. Data on sensitivity and dynamic range using kilohertz laser rates are also summarized. These results indicate that combinations of high‐performance MALDI‐TOF and TOF‐TOF with off‐line high‐capacity separations may ultimately provide throughput and dynamic range several orders of magnitude greater than those currently available with electrospray LC‐MS and MS‐MS. Copyright © 2009 John Wiley & Sons, Ltd.     

半乳糖修饰人血清白蛋白的质谱分析

研究发现,存在于哺乳动物肝细胞膜上的血浆无唾液酸糖蛋白受体(asialoglycoproteinreceptor,ASGP-R)是一种肝结合蛋白(hepaticbindingprotein,HBP),能专一性识别分子末端带有半乳糖残基的糖蛋白并与之结合,定向转到肝细胞内的溶酶体进行代谢,针对ASGP-R作为靶点的肝靶向药物载体半乳糖化人血白蛋白偶合物(galactosyl-neoglycoalbumin,NGA),已引起药物研究工作者的极大重视。将药物与带有半乳糖残基的蛋白偶合,可提高药物的肝靶向性,提高治疗指数,减少非靶器官的毒副作用。大量研究表明,肝结合蛋白受体介导的肝靶向给药系统是最具研究价值和应用前景的肝靶向给药系统之一。     

Combined techniques for the characterization of linear–hyperbranched hybrid poly(butylene adipate) copolymers

Linear–hyperbranched hybrid poly(butylene adipate) (HPBA) copolymers were synthesized through a branching reaction between the linear tailored prepolymer terminated with methyl ester groups and different mol percents of the 1,1,1‐tris(hydroxymethyl) propane (TMP) as branching agent, using the titanium(IV) isopropoxide as catalyst, at 180 °C under vacuum for different times. All samples were characterized by NMR and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI‐TOF MS). In particular, MALDI‐TOF mass spectra of the unfractionated and size exclusion chromatography (SEC)‐fractionated hyperbranched (HB) samples gave information on their composition, on the end groups as well as on the TMP units present in each family of HB macromolecules. HB chains containing cyclic branches and ether bonds formed by intermolecular transesterification and intramolecular and intermolecular transetherification side reactions, respectively, were also revealed by MALDI‐TOF MS analysis. All samples were also investigated by SEC. The average molar masses (MMs) evaluated by SEC calibrated with the polystyrene (PS) narrow standards were overestimated with respect to those calculated by the SEC/MALDI‐TOF MS self‐ calibration method, which gave reliable values. Moreover, it also showed that the hydrodynamic volume of the HPBA polymers was higher than that of the linear PSs with similar MMs. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Block length determination of the block copolymer mPEG‐b‐PS using MALDI‐TOF MS/MS

The molar mass determination of block copolymers, in particular amphiphilic block copolymers, has been challenging with chromatographic techniques. Therefore, methoxy poly(ethylene glycol)‐b‐poly(styrene) (mPEG‐b‐PS) was synthesized by atom transfer radical polymerization (ATRP) and characterized in detail not only by conventional chromatographic techniques, such as size exclusion chromatography (SEC), but also by matrix‐assisted laser/desorption ionization tandem mass spectrometry (MALDI‐TOF MS/MS). As expected, different molar mass values were obtained in the SEC measurements depending on the calibration standards (either PEG or PS). In contrast, MALDI‐TOF MS/MS analysis allowed the molar mass determination of each block, by the scission of the weakest point between the PEG and PS block. Thus, fragments of the individual blocks could be obtained. The PEG block showed a depolymerization reaction, while for the PS block fragments were obtained in the monomeric, dimeric, and trimeric regions as a result of multiple chain scissions. The block length of PEG and PS could be calculated from the fragments recorded in the MALDI‐TOF MS/MS spectrum. Furthermore, the assignment of the substructures of the individual blocks acquired by MALDI‐TOF MS/MS was accomplished with the help of the fragments that were obtained from the corresponding homopolymers. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

Tandem mass spectrometry of poly(ethylene imine)s by electrospray ionization (ESI) and matrix‐assisted laser desorption/ionization (MALDI)

In this contribution, linear poly(ethylene imine) (PEI) polymers, which are of importance in gene delivery, are investigated in detail by using electrospray ionization‐quadrupole‐time of flight (ESI‐Q‐TOF) and matrix‐assisted laser desorption/ionization‐time of flight (MALDI‐TOF) mass spectrometry (MS). The analyzed PEIs with different end groups were synthesized using the polymerization of substituted 2‐oxazoline via a living cationic ring‐opening polymerization (CROP) and a subsequent hydrolysis under acidic conditions. The main goal of this study was to identify linear PEI polymers in a detailed way to gain information about their fragmentation pathways. For this purpose, a detailed characterization of three different linear PEIs was performed by using ESI‐Q‐TOF and MALDI‐TOF MS in combination with collision‐induced dissociation (CID) experiments. In ESI‐MS as well as MALDI‐MS analysis, the obtained spectra of PEIs resulted in fitting mass distributions for the investigated PEIs. In the tandem MS analysis, a 1,2‐hydride shift with a charge‐remote rearrangement via a four‐membered cyclic transition state, as well as charge‐induced fragmentation reactions, was proposed as the main fragmentation mechanisms according to the obtained fragmentation products from the protonated parent peaks. In addition, heterolytic and homolytic cleavages were proposed as alternative fragmentation pathways. Moreover, a 1,4‐hydrogen elimination was proposed to explain different fragmentation products obtained from the sodiated parent peaks. Copyright © 2012 John Wiley & Sons, Ltd.     

Comprehensive chemical profiling of Guizhi Fuling capsule by the combined use of gas chromatography-mass spectrometry with a deconvolution software and rapid-resolution liquid chromatography quadrupole time-of-flight tandem mass spectrometry

Herbal formulations are complex natural mixtures. Researchers usually tend to focus more on analysis of nonvolatile components but pay less attention to volatile compounds. In this study, an analytical strategy combining two approaches was established for comprehensive analysis of herbal formulations. Guizhi Fuling capsule (GFC), a drug approved by the FDA to enter phase II clinical trial for treatment of primary dysmenorrhea, was taken as a case for analysis. Gas chromatography–mass spectrometry (GC‐MS) with automated mass spectral deconvolution and identification system (AMDIS) led to rapid identification of 48 volatile components including four acetophenones, three fatty acid esters, 13 phenylpropanoids and 19 sesquiterpenes. Most of them were found from Guizhi. The volatile oils of Guizhi have been proved to exhibit many pharmacological activities. This is helpful in understanding the pharmacological mechanism of GFC. Furthermore, AMDIS turned out to be efficient and reliable for analysis of complex herbal formulations. Rapid‐resolution liquid chromatography (RRLC) coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (ESI‐Q‐TOF MS/MS) allowed the identification of 70 nonvolatile components including six acetophenones, 12 galloyl glucoses, 31 monoterpene glycosides, three phenols and 12 triterpene acids. Fragmentation behaviors of assigned components, especially triterpene acids, which are hard to identify by low‐resolution MS, were first investigated by TOF MS/MS. Characteristic ions and typical loss of assigned triterpene acids were summarized. Combinatorial use of GC‐MS‐AMDIS and RRLC‐ESI‐Q‐TOF MS/MS could be of great help in global qualitative analysis of GFC, as well as other herbal products. Copyright © 2012 John Wiley & Sons, Ltd.     

Phosphorylation site localization in peptides by MALDI MS/MS and the Mascot Delta Score

Owing to its broad biological significance, the large-scale analysis of protein phosphorylation is more and more getting into the focus of proteomic research. Thousands of phosphopeptides can nowadays be identified using state-of-the-art tandem mass spectrometers in conjunction with sequence database searching, but localizing the phosphate group to a particular amino acid in the peptide sequence is often still difficult. Using 180 individually synthesized phosphopeptides with precisely known phosphorylation sites (p-sites), we have assessed the merits of the Mascot Delta Score (MD score) for the assignment of phosphorylation sites from tandem mass spectra (MS/MS) generated on four different matrix-assisted laser desorption ionization (MALDI) mass spectrometers including tandem time-of-flight (TOF/TOF), quadrupole time-of-flight, and ion trap mass analyzers. The results show that phosphorylation site identification is generally possible with false localization rates of about 10%. However, a comparison to previous work also revealed that phosphorylation site determination by MALDI MS/MS is less accurate than by ESI-MS/MS particularly if several and/or adjacent possible phosphorylation acceptor sites exist in a peptide sequence. We are making the tandem MS spectra and phosphopeptide collection available to the community so that scientists may adapt the MD scores reported here to their analytical environment and so that informatics developers may integrate the MD score into proteomic data analysis pipelines.     

Evaluation of automated single mass spectrometry to tandem mass spectrometry function switching for comprehensive drug profiling analysis using a quadrupole time-of-flight mass spectrometer

A liquid chromatographic mass spectrometric strategy for systematic toxicological analysis (STA) is presented using the automatic 'on-the-fly' single mass spectrometry mode to tandem mass spectrometry mode (MS to MS/MS) switching abilities of a quadrupole time-of-flight (Q-TOF) instrument. During the chromatographic run, the quadrupole is initially set to transmit all masses until (an) ion(s) reaches a certain set threshold. Thereupon, the quadrupole automatically switches to the MS/MS mode, selecting the ion(s), which are subsequently fragmented in the high-efficiency hexapole collision cell, thus generating product ions that are further mass analyzed by the TOF. By limiting the TOF spectral accumulation time in the MS/MS mode to a statistically acceptable minimum, the quadrupole almost instantly switches back to the MS mode. Qualitative information, comprising the complementary MS ([M + H](+) ion mass) and MS/MS (informative product ion profile) data, as well as quantitative information obtained by integration of the MS extracted ion chromatogram(s), can be obtained in one single acquisition. Optimization of the automatic switching parameters, such as threshold, TOF spectral accumulation time, detection window and collision energy, was carried out by injection of a mix of 17 common drugs which were not necessarily baseline separated in the chromatographic system used. Indeed, the complete separation of the drugs is not deemed necessary since up to 8 different ions can 'simultaneously' be selected for MS/MS if they reach the preset criteria. In addition, the quantitative performance of the method was defined. In a second phase, the developed method was field-tested. To that end, the resulting data from extracts of urine samples were compared with and found to be in close concordance with those obtained by a standard toxicological analysis. This innovative approach clearly holds the potential for a substantial advance in the introduction of LC/MS in STA.     

A comparative study of the fragmentation of neutral lactooligosaccharides in negative-ion mode by UV-MALDI-TOF and UV-MALDI ion-trap/TOF mass spectrometry

Structure analyses of underivatized neutral lacto oligosaccharides are systematically performed by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI TOF MS) and UV-MALDI ion-trap time-of-flight mass spectrometry (ion-trap/TOF MS) acquired in negative-ion mode. Interestingly, their fragmentation significantly differ each other. In postsource decay (PSD) in UV-MALDI TOF MS, cross-ring cleavage at the reducing terminal predominates. On the other hand, glycosyl bond cleavage (C-type fragmentation) takes place preferentially in collision induced dissociation (CID) in UV-MALDI ion-trap/TOF MS. The cross-ring cleavage in PSD similar to that in in-source decay occurs via a prompt reaction path characteristic of the UV-MALDI process itself. The product ion spectra of UV-MALDI ion-trap/TOF MS are similar to the electrospray ionization (ESI) ion-trap or quadrupole/TOF CID product ion spectra. During ion-trap/TOF MS experiments, the deprotonated molecular ions survive for several tens of milliseconds after CID event because the high internal energy chlorinated precursor ions are cooled by collisional cooling in the ion trap. The results obtained suggest that the PSD from the chlorinated precursor ion in UV-MALDI TOF MS might proceed as a two-step reaction; in the first, a high internal energy deprotonated molecular ion is generated as a reaction intermediate during the flight in the drift tube, and in the second, the rapid decomposition from the deprotonated molecular ion takes place.     

MALDI TOF/TOF tandem mass spectrometry as a new tool for amino acid analysis

This is the first report of an application of collisionally induced fragmentation of amino acids (AA) and their derivatives by MALDI TOF/TOF tandem mass spectrometry (MS). In this work, we collected the data on high-energy fragmentation reactions of a large group of protonated amino acids and their derivatives with the goal of determining which product ions are analyte specific and if yields of these fragment could be used for quantitative analysis. From 34 different amino acids (20 alpha-amino acids, beta-amino acids, homocysteine, GABA, and modified AA Met sulfone and sulfoxide, hydroxyproline, etc.) we observed that high yields of the target specific immonium ions and fragmentation patterns are most similar to EI or FAB CID on sector instruments. The major exceptions were two highly basic amino acids, Arg and Orn. It is noted that neither beta-, gamma-, nor delta-amino acids produce immonium ions. As might be predicted from high-energy CID work on peptides from the sectors and TOF/TOF, the presence of specific indicator ions in MALDI tandem MS allows distinguishing isomeric and isobaric amino acids. These indicator ions, in combination with careful control of data acquisition, ensure quantitative analysis of amino acids. We believe our data provide strong basis for the application of MALDI TOF/TOF MS/MS in qualitative and quantitative analysis of amino and organic acids, including application in clinical medicine.     

Modeling bacteriophage amplification as a predictive tool for optimized MALDI‐TOF MS‐based bacterial detection

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) is a valuable tool for rapid bacterial detection and identification but is limited by the need for relatively high cell count samples, which have been grown under strictly controlled conditions. These requirements can be eliminated by the natural infection of a viable bacterial species of interest with a host‐specific phage. This produces a rapid increase in phage protein concentrations in comparison to bacterial concentrations, which can in turn be exploited as a method for signal amplification during MALDI‐TOF MS. One drawback to this approach is the requirement for repetitive, time‐consuming sample preparation and analysis applied over the course of a phage infection to monitor phage concentrations as a function of time to determine the MALDI‐TOF MS detection limit. To reduce the requirement for repeated preparation and analysis, a modified phage therapy model was investigated as a means for predicting the time during a given phage infection when a detectable signal would occur. The modified model used a series of three differential equations composed of predetermined experimental parameters including phage burst size and burst time to predict progeny phage concentrations as a function of time. Using Yersinia pestis with plague diagnostic phage ?A1122 and Escherichia coli with phage MS2 as two separate, well‐characterized model phage–host pairs, we conducted in silico modeling of the infection process and compared it with experimental infections monitored in real time by MALDI‐TOF MS. Significant agreement between mathematically calculated phage growth curves and those experimentally obtained by MALDI‐TOF MS was observed, thus verifying this method's utility for significant time and labor reduction. Copyright © 2012 John Wiley & Sons, Ltd.     

Tandem mass spectrometric accurate mass performance of time-of-flight and Fourier transform ion cyclotron resonance mass spectrometry: a case study with pyridine derivatives

The interpretation of mass spectra is a key process during compound identification, and the combination of tandem mass spectrometry (MS/MS) with high-accuracy mass measurements may deliver crucial information on the identity of a compound. Obtaining accurate mass data of fragment ions in MS/MS reveals the particular problem of mass calibration when a lockmass, which is frequently used to obtain accurate masses in MS, is absent. An alternative technique is to recalibrate the MS/MS spectrum using a reference MS/MS spectrum acquired under the same conditions. We have tested and validated this approach using a hybrid quadrupole/orthogonal acceleration reflectron-type time-of-flight (TOF) mass spectrometer. The results were compared with those obtained under similar conditions on a Fourier transform ion cyclotron resonance (FT-ICR) instrument. We found that the mass accuracy observed with such an "external" recalibration on the TOF instrument in MS/MS is identical to what can be obtained on a similar instrument operating in one-dimensional MS mode using the lockmass technique. However, mass accuracy in both cases is one order of magnitude inferior to that obtained using FTMS, and also inferior to that observed using sector field MS when operated at comparable resolution. Nevertheless, for small (<200 Da) molecules, this mass accuracy was still sufficient to have the "true" elemental composition identified as the first hit in about 70% of all cases. It was possible to elucidate the fragmentation mechanism of eight azaheterocycles containing a pyridine moiety, where the accurate mass data from the TOF instrument allowed distinction between two alternative fragmentation pathways.     

Comparison of three types of mass spectrometers for HPLC/MS analysis of perfluoroalkylated substances and fluorotelomer alcohols

The performance of three different types of mass spectrometers (MS) coupled to high performance liquid chromatography (HPLC) was compared for trace analysis of perfluoroalkylated substances (PFAS) and fluorotelomer alcohols (FTOHs). Ion trap MS in the full scan and product ion MS2 mode, time-of-flight (TOF) high resolution MS and quadrupole MS in the selected ion mode as well as triple quadrupole tandem MS were tested. Electrospray ionisation in the negative ion mode [ESI-] was best suited for all instruments and compounds. PFAS could only be separated by a buffered mobile phase, but the presence of buffer suppressed the ionisation of FTOHs. Therefore, two independent chromatographic methods were developed for the two compound classes. Mass spectra and product ion spectra obtained by in-source and collision induced dissociation fragmentation are discussed including ion adduct formation. Product ion yields of PFAS were only in the range of 0.3 to 12%, independent from the applied MS instrument. Ion trap MS2 gave product ion yields of 20 to 62% for FTOHs, whereas only 4.1 to 5.8% were obtained by triple quadrupole tandem MS. Ion trap MS was best suited for qualitative analysis and structure elucidation of branched isomeric structures of PFAS. Providing typical detection limits of 5 ng injected in MS2 mode, it was not sensitive enough for selective trace amount quantification. TOF high resolution MS was the only technique combining high selectivity and excellent sensitivity for PFAS analysis (detection limits of 2 to 10 pg), but lacked the possibility of MS-MS. Triple quadrupole tandem MS was the method of choice for quantification of FTOHs with detection limits in the low pg range. It is also well suited for the determination of PFAS, though its detection limits of 10 to 100 pg in tandem MS mode are about one order of magnitude higher than for TOF MS.     

The identification of synthetic homopolymer end groups and verification of their transformations using MALDI‐TOF mass spectrometry

Recent advances in the resolving power of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) enable the detailed characterization of linear homopolymers, and in particular provide invaluable data for the determination of their end‐group functionalities. With the growing importance of macromolecular coupling reactions in building complex polymer architectures, the ability to accurately monitor end‐group transformations is becoming increasingly important for synthetic polymer chemists. This tutorial demonstrates the application of MALDI‐TOF MS in determining both end‐group functionalities and their transformations for linear homopolymers. Examples of both polycaprolactone and polystyrene are examined, and the strengths and weaknesses of various approaches to data analysis are given. Copyright © 2010 John Wiley & Sons, Ltd.     

An introduction to quadrupole-time-of-flight mass spectrometry

A brief introduction is presented to the basic principles and application of a quadrupole-time-of-flight (TOF) tandem mass spectrometer. The main features of reflecting TOF instruments with orthogonal injection of ions are discussed. Their operation and performance are compared with those of triple quadrupoles with electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) TOF mass spectrometers. Examples and recommendations are provided for all major operational modes: mass spectrometry (MS) and tandem MS (MS/MS), precursor ion scans and studies of non-covalent complexes. Basic algorithms for liquid chromatography/MS/MS automation are discussed and illustrated by two applications.     

Mass spectrometric characterization of low-molecular-mass color pI markers and their use for direct determination of pI value of proteins

The use of low-molecular-mass color pI markers for the determination of pI values of proteins in gel isoelectric focusing (IEF) in combination with mass spectrometry is described. Different types of substituted phenols of known pI values within the mass range 250-400 were used here as pI markers. The pure, synthesized pI markers were studied by MALDI-TOF/TOF MS. Fragmentation studies of the pI markers were also performed. Only stable and well-characterized pI markers were used in this work. The selected pI markers were mixed with proteins, deposited on a gel and separated in a pH gradient. Color pI markers enable supervision of progress of the focusing process and also estimation of the position of the invisible focused bands. The separated bands of the pI markers (containing separated proteins) were excised, and the pI markers were eluted from each gel piece by water/ethanol and identified by MALDI-TOF/TOF MS. From the washed gel pieces the remaining carrier ampholytes were then washed out and proteins were in-gel digested with trypsin. The obtained peptides were measured by MALDI-TOF/TOF MS and the proteins identified via a protein database search. This procedure allows avoiding time-consuming protein staining and destaining procedures, which shortens the analysis time roughly by half. For comparison, IEF gels were stained with Coomassie Brilliant Blue R 250 and proteins in the gel bands were identified according to the standard proteomic protocol. This work has confirmed that our approach can give information about the correct pI values of particular proteins and shorten significantly the time of analysis.     

Isotope tag method for quantitative analysis of carbohydrates by liquid chromatography-mass spectrometry

We have previously demonstrated that liquid chromatography/mass spectrometry equipped with a graphitized carbon column (GCC-LC/MS) is useful for the structural analysis of carbohydrates in a glycoprotein. Here, we studied the monosaccharide composition analysis and quantitative oligosaccharide profiling by GCC-LC/MS. Monosaccharides were labeled with 2-aminopyridine and then separated and monitored by GCC-LC/MS in the selective ion mode. The use of tetradeuterium-labeled pyridylamino (d4-PA) monosaccharides as internal standards, which were prepared by the tagging of standard monosaccharides with hexadeuterium-labeled 2-aminopyridine (d6-AP), afforded a good linearity and reproducibility in ESIMS analysis. This method was successfully applied to the monosaccharide composition analysis of model glycoproteins, fetuin, and erythropoietin. For quantitative oligosaccharide profiling, oligosaccharides released from an analyte and a standard glycoprotein were tagged with d0- and d6-AP, respectively, and an equal amount of d0- and d4-PA oligosaccharides were coinjected into GCC-LC/MS. In this procedure, the oligosaccharides that existed in either analyte or a standard glycoprotein appeared as single ions, and the oligosaccharides that existed in both analyte and a standard glycoprotein were detected as paired ions. The relative amount of analyte oligosaccharides could be determined on the basis of the analyte/internal standard ion-pair intensity ratio. The quantitative oligosaccharide profiling enabled us to make a quantitative and qualitative comparison of glycosylation between the analyte and standard glycoproteins. The isotope tag method can be applicable for quality control and comparability assessment of glycoprotein products as well as the analysis of glycan alteration in some diseases.