Direct Infusion ESI-MS Research Articles

Direct glycosylation analysis of intact monoclonal antibodies combining ESI MS of glycoforms and MALDI-in source decay MS of glycan fragments

Monoclonal antibody (mAb) glycoengineering has the potential to improve the efficacy of biopharmaceuticals by fine-tuning specific biological properties. Glycosylation analysis is key to monitoring the glycoengineering process. Various mass spectrometry (MS)-based methods are available to characterize mAb glycosylation at different structural levels, but comprehensive analysis is typically time-consuming and costly. Here, we present an approach that combines conventional intact mass measurement of glycoforms by direct infusion ESI-MS with an advanced MALDI-in-source decay FT-ICR MS method for direct analysis of glycans in intact mAbs, without the need for enzymatic release and separation. Using a sodium-doped MALDI matrix, glycans were directly released as ISD fragment ions from the intact mAbs during the ionization process. Measurement of 0,2A fragment signals yielded reproducible glycan profiles that were consistent with conventional methods, yet was achieved with unprecedented speed, providing complementary information to that obtained through intact mass measurement. The methods were applied to standard and glycoengineered trastuzumab and rituximab, allowing rapid glycosylation profiling and structural analysis of glycans by tandem MS of selected ISD fragment ions. This fast approach can facilitate the early-phase development of glycoengineering processes by constraining further in-depth analyses. We envision a broader applicability in studies focused on glycosylation changes in mAbs.

Lipid Profile Determination by Direct Infusion ESI-MS and Fatty Acid Composition by GC-FID in Human Milk Pools by Folch and Creamatocrit Methods

Human milk (HM) is extremely important to the baby, containing a varied and balanced range of nutrients fundamental to the ideal development of the newborn. Among these nutrients, HM lipids provide 40-60% of the energy. In HM banks, the creamatocrit method is employed as a quick and solvent-free tool, however, since Folch methodology is considered reference for lipid extraction, this study compared the fatty acid (FA) composition and the lipid profile by gas chromatography with flame ionization detector (GC-FID) and direct infusion by electrospray ionization mass spectrometry (DI-ESI-MS) in pools of different HM phases, obtained from Folch and creamatocrit methods, in order to elucidate which method provides better information about HM lipids. Lipid compositon results by Folch were superior to the results obtained by creamatocrit in HM pools investigated. It means, the lipid profiles of all HM pools were more intense, and the number of FA identified by Folch was also higher in comparison to the creamatocrit method (21 and 6 FAs, respectively). GC-FID analysis in combination with principal component analysis (PCA) divided the methods, confirming a greater contribution of the Folch method. Therefore, Folch methodology revealed an effective lipid extraction in comparison to creamatocrit method.

Cysteinylated and glycated human serum albumin levels are increased in human heart failure

Cysteinylated human serum albumin (Cys-HSA) is a post-translational modification of human serum albumin (HSA) where the sulfhydryl group (SH) of cysteine in position 34 (Cys34) becomes oxidized by a cysteine [1] and this oxidation easily occurs when exposed to oxidative stress [2]. The degree of oxidized Cys34 in HSA is correlated with oxidative stress related pathological conditions [3]. Indeed, increased Cys-HSA levels have been detected in patients undergoing end stage renal disease [4], coronary artery disease [2], chronic liver disease or diabetes mellitus [3] as well as in pregnant women with intrauterine growth restriction [5]. Functional loss of HSA due to post-translational modification could influence homeostasis, which may contribute to the progression of chronic diseases. For example, Oettl et al. reported that, in advanced liver disease, oxidative damage impairs the binding properties of HSA resulting in increased tissue distribution of toxic endogenous compounds, and thus enhancing the risk of tissue damage related to complications [6]. Similarly, the loss of significant oxidant buffering capacity of HSA due to Cys-HSA may lead pregnancy complications such as intrauterine growth restriction [5]. In this study we examined for the first time the extent of Cys-HSA in plasma of patients with heart failure [NYHA class III and IV (n class III =10, n class IV = 10)] compared with healthy subjects (n=11) by using a direct infusion ESI-MS. Blood samples were collected in citrate tubes and centrifuged immediately after collection. Plasma fraction was stored at -80 °C. Before the analysis, plasma samples were diluted 1:200 in water containing 50% acetonitrile and 0.1% formic acid and infused into a triple quadrupole mass spectrometer (Xevo TQ-S from Waters). The relative content of the HSA isoforms (Cys-HSA and glycated-HSA) were determined by measuring their relative intensities. Results showed a significant increase of Cys-HSA in heart failure (HF) patients (13.11%±5 and 17.31%±3.6 for NYHA III and IV, respectively) with respect to age-matched healthy subjects (9.87%±3). Furthermore, the levels of glycated-HSA were higher in HF patients (7.72%±1.49 and 8.04%±0.85, for NYHA III and IV, respectively) in comparison with age-matched healthy subjects (6.535%+0.54). Finally, HF patients showed a significant decrease in the total level of HSA as expected [7]. In conclusion, this study revealed an increased level of cysteinylation and glycation of HSA in patients with HF expanding the current knowledge that different residues on HSA can undergo certain post-translational modifications in specific environments in oxidative stress-related diseases. Future studies will establish the utility of monitoring the redox status of Cys34 in HSA as a marker for oxidative stress in HF and its potential role in the onset and progression of the disease. Finally, since HSA has an established role as a blood stream carrier, it will be interesting and exciting to test how these modifications can affect drug delivery of various pharmacological treatments in HF.

Detecting lipoxidation: diagnostic tools for the translation and commercial application

Oxidative stress is strongly associated with the pathophysiology of inflammation diseases such as sepsis, diabetes and cardiovascular diseases. The oxidants formed in inflammation can cause oxidative damage to lipids, producing short chain electrophilic oxidized lipid products that can react with nucleophilic amino acid residues of proteins, a process known as lipoxidation [1]. 4-hydroxy-2-nonenal (HNE) is one of the quantitatively most important products of lipid peroxidation and can react by Michael addition or form Schiff base adducts with proteins, leading to cellular dysfunction. As lipoxidation products have potential as biomarkers of oxidative stress and disease, development of rapid assays for them is required. The aim of this work was to produce antibodies that recognize HNE-modified human serum albumin for future use in diagnostic assays of plasma. Human serum albumin (HSA), the most abundant protein of the human sera, was reacted with HNE at a 1:10 ratio. Analysis of intact HSA-HNE by direct infusion ESI-MS suggested the presence of (on average) 4.7 molecules of HNE/HSA and MALDI-TOF analysis showed an increase in mass corresponding to the addition of 3 molecules of HNE/HSA. Proteomic analysis of tryptic digests gave with confident identification only of HNE Michael addition, on the histidine of peptide VH242TECCHGDLLECADDR. To complement the results, an ELISA assay was performed with a commercial anti-HNE antibody and specific binding to HSA-HNE was observed, in contrast to low binding of un-modified HSA. The HSA-HNE adducts were subsequently immunized into sheep for generation of polyclonal antibodies and ELISA assays performed with enriched antibodies showed specific binding to HNE-modified proteins. In parallel, a potential reactive peptide sequence (LQQC34PFE; m/z= 862.3) from HSA was synthesised and reacted with HNE. Using ESI-MS, HNE Michael adducts (m/z= 1018.4) on the cysteine residue were confirmed and Schiff base adducts (m/z= 1000.4), probably on the free N-terminal group, were also observed. These adducts were purified by reverse phase HPLC and immunized in rabbit and sheep for generation of antibodies anti-HNE-modified LQQCPFE. In conclusion, HNE adducts on HSA were confirmed and used for the generation of polyclonal antibodies. Enriched antibodies showed binding to HNE-modified HSA, but some cross-reactivity to HSA. Future work includes the improvement of the antibody purification process and testing anti-sera from rabbit and sheep immunized with LQQC(HNE)PFE.

Investigation by direct-infusion ESI–MS and GC–MS of an alleged Leuckart route-specific impurity of methamphetamine

Impurity profiling has been used as a useful tool for analyzing nearly every drug class currently known on the illicit market. Impurities present within seized samples have the potential to determine source of origin, route of synthesis used, as well as provide a useful clue into the potential reaction mechanisms that are present for each synthetic procedure. Perhaps the most well studied of these impurity profiles exists for methamphetamine, including information to more than one route of synthesis. Within the present study, a complete synthesis of methamphetamine was performed, including a reductive amination of phenylpropanone (P2P) using methylamine hydrochloride and sodium triacetoxyborohydride (STAB) rather than the conventional aluminum mercury amalgam commonly found in the literature. During the analysis of the final product from this reaction, a major impurity within the reaction, bis(1-phenylpropan-2-yl)amine (m/z 253), was detected by GC–MS as well as direct-infusion ESI–MS. This impurity has been previously reported as a Leuckart route-specific impurity. Its detection within the reductive amination of P2P points towards the use of impure methylamine hydrochloride containing some traces of acid, and provides further insight into the reductive amination of P2P. In both the Leuckart reaction and this reductive amination via STAB, the presence of acid and ammonia leads to this impurity.

Structural Characterization and Disulfide Assignment of Spider Peptide Phα1β by Mass Spectrometry.

Native Phα1β is a peptide purified from the venom of the armed spider Phoneutria nigriventer that has been shown to have an extensive analgesic effect with fewer side effects than ω-conotoxin MVIIA. Recombinant Phα1β mimics the effects of the native Phα1β. Because of this, it has been suggested that Phα1β may have potential to be used as a therapeutic for controlling persistent pathological pain. The amino acid sequence of Phα1β is known; however, the exact structure and disulfide arrangement has yet to be determined. Determination of the disulfide linkages and exact structure could greatly assist in pharmacological analysis and determination of why this peptide is such an effective analgesic. Here, we used biochemical and mass spectrometry approaches to determine the disulfide linkages present in the recombinant Phα1β peptide. Using a combination of MALDI-MS, direct infusion ESI-MS, and nanoLC-MS/MS analysis of the undigested recombinant Phα1β peptide and digested with AspN, trypsin, or AspN/trypsin, we were able to identify and confirm all six disulfide linkages present in the peptide as Cys1-2, Cys3-4, Cys5-6, Cys7-8, Cys9-10, and Cys11-12. These results were also partially confirmed in the native Phα1β peptide. These experiments provide essential structural information about Phα1β and may assist in providing insight into the peptide's analgesic effect with very low side effects. Graphical Abstract ᅟ.

18-Crown-6 spiking in direct infusion ESI–MS analysis of complex mixtures: “One ion per analyte” relationship facilitating ion assignments and eliminating isobaric interferences

Spiking with 18-crown-6/trifluoracetic acid is shown to sequester metal cations thus eliminating unwanted [TAG+Na]+ and [TAG+K]+ ions whereas favoring analyte detection as sole [TAG+H]+ ions in the ESI(+)-MS analysis of methanolic solutions of soybean oil. This sequestering eliminates ion splitting and its resulting isobaric interferences, thus increasing spectra quality greatly facilitates analyte identification. This “one ion per analyte relationship” is highly beneficial for direct infusion analysis of complex mixtures by ESI–MS.

Wood chemotaxonomy via ESI-MS profiles of phytochemical markers: the challenging case of African versus Brazilian mahogany woods

Chemotaxonomy via direct infusion ESI-MS was proposed as a rapid and simple method to differentiate the Brazilian and African mahogany, which has been used for Amazon reforestation.

Direct infusion ESI-MS analysis for metabolite profiling of human plasma using various fractionation techniques.

Metabolomics is a real challenge owing to the complexity and chemical heterogeneity of biological samples. In this study, a comparative analysis of metabolite profiling using ten metabolite extraction techniques were investigated on a pooled plasma sample followed by direct infusion ESI-MS analysis on both positive and negative modes. Methodology & Results: Metabolites from a pooled sample of 50 healthy volunteers were separated one- and two-dimensionally utilizing solvent precipitation, solid-phase extraction and molecular weight fractionation. Numbers of unique metabolites that are specific to a particular sample preparation approach were also identified by online available database, Metlin. The (1)H NMR study of different extraction procedures were also recorded to elaborate the comparative profiling of the various fractionation procedures. The results of this study suggest that the metabolite extraction procedures based on 1- and 2-dimensions, followed by direct infusion ESI-MS analysis were able to detect endogenous metabolites belonging to different chemical classes.

Extractive electrospray ionization mass spectrometry of ionic liquids

The growing role of ionic liquids (ILs) in industry and research is creating an urgent need for the development of analytical methods for the reliable characterization and control of the composition of the ILs. The application of mass spectrometry (MS) to the analysis of ILs is limited by the poor tolerance of classical MS methods toward samples with high salt concentrations and viscosity. Herein, extractive electrospray ionization mass spectrometry (EESI-MS) was applied to directly analyze various room temperature ILs such as 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF6]) 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF4]), and 1-butylpyridinium tetrafluoroborate ([BuPy][BF4]). The EESI mass spectra of ILs diluted in methanol revealed great similarities to the mass spectra of the same samples assessed by direct infusion ESI-MS. Both cationic (C+) and anionic (A−) IL components could be detected with a high sensitivity from strongly diluted samples (<10−7 M). [CA − H]− or [CA + H]+ signals were observed for certain ILs using EESI-MS, and in good agreement with their classification based on the acid–base theory. With only minor alterations to the experimental setup, analysis of the ILs could be performed without dilution, which allowed a direct observation of the IL aggregates in the gas phase at a speed of >2 samples per min. EESI-MS allows the direct analysis of ILs in a broad concentration range with a high tolerance to chemical contamination. The reported examples suggest the potential for EESI-MS to interrogate large IL assays, which is critical in both fundamental physical chemistry studies and industrial applications.

Comparative core fucosylation analysis of some major therapeutic antibody N‐glycans by direct infusion ESI‐MS and CE–LIF detection

The analysis of carbohydrate moieties of glycoprotein biopharmaceuticals is of high importance, especially because glycosylation changes can impact the biological effect of the drugs. In the case of monoclonal antibody therapeutics, the degree of core fucosylation significantly influences its effector function, thus should be closely monitored during the clone selection, development and manufacturing processes. MS analysis of labile sugar residues such as core fucosylation and terminal silylation may be misleading as such residues can break off during the ionization process or alter the ionization efficiency, both of which affect the results. In the case of monoclonal antibody therapeutics tailored for antibody-dependent cellular cytotoxicity function, core fucosylation should be kept minimal; therefore, close attention should be paid to the prevalence of this particular sugar residue. This paper reports on the core fucosylation analysis of some major immunoglobulin G N-glycans by direct infusion ESI-MS and CE-LIF. In comparison to the industry standard of CE-LIF, a lower degree of core fucosylated structures was found by ESI-MS analysis, emphasizing the need to use orthogonal quantitative separation methods in addition to MS.

Electrochemical Oxidation of Ethinylestradiol on a Commercial Ti/Ru0.3 Ti0.7O2 DSA Electrode

The electrochemical oxidation of the hormone ethinylestradiol in an aqueous-methanolic medium by the application of a constant current of 40 mA cm−2 in a flow cell with a commercial Ti/Ru0.3 Ti0.7O2 electrode was evaluated. The effect caused by the use of NaCl as a support electrolyte was also investigated. Hence, HPLC-UV analyses revealed that ethinylestradiol was almost totally consumed after a 60 min reaction time in the presence of NaCl. Conversely, much lower degradation rates were obtained when NaCl was not employed. Moreover, direct infusion ESI-MS and GC-MS analysis revealed that apparently no degradation products had been formed under these conditions. Hence, this study clearly demonstrated that such electrochemical treatment can be efficiently used to promote the complete degradation (and probably mineralization) of the hormone ethinylestradiol.

On-line monitoring of stevioside sweetener hydrolysis to steviol in acidic aqueous solutions

Abstract Stevioside, a potent sweetener obtained from leaves of Stevia rebaudiana (Bertoni), is the glycone of steviol. However, despite its natural origin, there has been concern about stevioside toxicity due to hydrolysis to the carcinogenic steviol. To approve it as an additive, the FAO/WHO committee on food additives has required further information about hydrolytic stability of the steviol glycoside in acidic foods and beverages. In this study, aqueous solutions of stevioside at different pH values were monitored in real time by direct infusion ESI-MS. Owing to the high speed and sensitivity of ESI-MS monitoring, fast hydrolysis of the stevioside molecule to steviol in aqueous acidic solutions was observed, particularly in acidic juices.

Fast Screening and Secure Confirmation of Milk Powder Adulteration with Maltodextrin via Electrospray Ionization−Mass Spectrometry [ESI(+)−MS] and Selective Enzymatic Hydrolysis

Direct-infusion electrospray ionization-mass spectrometry [ESI(+)-MS] of several milk powder samples, confiscated by the Brazilian Federal Police, showed ions accounting for sodiated and potassiated molecules of disaccharides (m/z 365 and 381) as well as trisaccharides (m/z 527 and 543), whereas monosaccharide ions were not detected. The trisaccharide ions were not detected in samples of genuine milk powder, raising the suspicion that their presence indicates adulteration by the addition of maltodextrin. In control samples, maltose and maltotriose were hydrolyzed by alpha-glucosidase and not beta-galactosidase, whereas lactose was resistant to alpha-glucosidase but was hydrolyzed with beta-galactosidase. Samples suspected of being adulterated behaved in the same fashion, confirming the presence of maltose and maltotriose or maltodextrin. Direct-infusion ESI-MS is shown therefore to provide rapid screening of milk powder for adulteration with maltodextrin, whereas its combination with selective enzymatic hydrolysis provides highly reliable confirmation for unambiguous results.

CZE and ESI-MS of Borate-Sugar Complexes

Capillary zone electrophoresis (CZE) with electrospray ionization (ESI) mass spectrometry (MS) was used to study borate (B−1) and sugar (L) complexes (LxB−1). Boric acid was adjusted to pH 10 with ammonium hydroxide to create an ESI-MS compatible CZE background electrolyte. We show for the first time that the electrophoretic peaks for each injected sugar contained both the substrates (i.e., sugar and/or multimers) and products (i.e., LxB−1). The effects of sheath liquid, temperature, and borate concentration were studied. The molecular mass information obtained from the ESI-MS provided new evidence on the mechanisms of borate-sugar complexation. Direct infusion ESI-MS and CZE-ESI-MS experiments strongly suggest that the formation of LxB−1 was from the direct reaction of a sugar or sugar multimer (Lx) and B−1. Larger LxB−1, where x > 2 were observed. Separation in the CZE dimension allows for the simultaneous analysis of a sugar mixture and simplified the ESI-MS analysis of sugars of the same molecular mass. The increase in sugar electrophoretic mobility caused by the increase in borate concentration was discussed in terms of the formation of LxB−1 complexes. In addition, the separation of five nucleosides by CZE using a borate electrolyte and detection using ESI-MS is demonstrated.

A new class of anthocyanin‐procyanidin condensation products detected in red wine by electrospray ionization multi‐stage mass spectrometry analysis

In our previous work, we have identified, in a model wine solution containing malvidin 3-glucoside, epicatechin and acetaldehyde, a new condensation product--hydroxylethyl-malvidin-3-glucoside-ethyl-epicatechin. The objective of this work was to verify the presence of such new condensation products in red wine. For this purpose, red wine was fractionated into various fractions by column chromatography on LiChroprep RP 18 and on Toyopearl 40 (F). The phenolic composition of each fraction was verified by HPLC-DAD and direct-infusion ESI-MS(n) analysis. In addition to the well-known anthocyanins and their acetyl and coumaroyl derivatives, and several direct and indirect anthocyanin-(epi)catechin condensation products, a new class of pigmented products, namely hydroxyethyl-anthocyanin-ethyl-flavanol compounds, have been detected in red wine. The new class of pigmented products would be expected to be the major pigments responsible for the color of aged red wine.

Polytungstate analysis by SEC-ICP-MS and direct-infusion ESI-MS

Polytungstate analysis by SEC-ICP-MS and direct-infusion ESI-MS

Safety assessment of food and herbal products containing hepatotoxic pyrrolizidine alkaloids: interlaboratory consistency and the importance ofN‐oxide determination

Two recent mass spectrometry-based reports concerning Senecio scandens yielded remarkably dissimilar pyrrolizidine alkaloid constituents. In both studies, and in a related analysis of Senecio scandens and Tussilago farfara using micellar electrokinetic chromatography, the presence of hazardous N-oxides of the alkaloids was either not considered or was inadequately considered. This raises concerns about the effectiveness of the methodologies used in these, and similar, studies in assessing the pyrrolizidine alkaloid content and the safety of food, food supplements and medicines for human use. To highlight essential analytical requirements for confident assessment of pyrrolizidine alkaloid-related safety of food and herbal products for human use. Direct infusion-ESI MS and HPLC-ESI MS were used to analyse samples derived from liquid-liquid partitioning experiments and from strong cation exchange, solid-phase extraction of pyrrolizidine alkaloids and their N-oxides. A simple solvent partitioning experiment using pure senecionine and senecionine-N-oxide, two constituents reported in one of the mass spectrometry-based studies of S. scandens, clearly demonstrated the inadequacy of the reported method to detect and quantitate hazardous pyrrolizidine alkaloid N-oxide components. A preliminary LCMS analysis of commercially-prepared extracts of comfrey roots (Symphytum officinale and S. uplandicum s. l.) was used as a model to highlight the analytical importance of N-oxides in the safety assessment of pyrrolizidine alkaloid-containing medicinal herbs. This study highlighted significant differences in the reported identification of pyrrolizidine alkaloids from the same plant species, and clearly demonstrated the inadequacy of some procedures to include N-oxides in the assessment of pyrrolizidine alkaloid-related safety of food and herbal products.

Identification of Oxidation Products of Squalene in Solution and in Latent Fingerprints by ESI-MS and LC/APCI-MS

An investigation was carried out to identify oxidation products of squalene (SQ) in latent fingerprints. Oxidation products of SQ incubated in solution with Rose Bengal as a photooxidizer were isolated by semipreparative HPLC-UV and identified by direct infusion ESI-MS and flow injection APCI-MS. Squalene hydroperoxides ranging from squalene monohydroperoxide (SQ-[OOH]) to SQ-[OOH]5 were identified together with SQ epoxide. SQ-[OOH] was the main oxidation product. An LC/APCI-MS method was developed and used to monitor the fate of SQ in solution and in latent fingerprints and the formation of SQ-[OOH] and SQ epoxide. SQ-[OOH] and SQ epoxide were detected in freshly deposited prints but increased markedly after 1 day and continued to increase up to 5 days after print deposition. By day 7, these substances could no longer be detected in prints. SQ was rapidly depleted from prints such that by day 7 it was no longer detected. A similar pattern was seen for SQ stored in the light in dichloromethane but with a slower formation of SQ-[OOH] and SQ epoxide. The oxidation of SQ in solution in the presence and absence of photooxidizer was shown by TLC to proceed as follows: SQ-->SQ-[OOH]+SQ epoxide. SQ-[OOH]-->SQ-[OOH]2-->SQ-[OOH]3-->SQ-[OOH]4+SQ-[OOH]5, with oxidation being more rapid in the presence of photooxidizer. SQ-[OOH]4 and SQ-[OOH]5 could still be detected at 20 days in a solution of SQ aged in solution in the absence of photooxidizer. The oxidation products of SQ should make suitable targets for development of new reagents for visualizing latent fingerprints in forensic science.

A comparison of direct infusion MS and GC‐MS for metabolic footprinting of yeast mutants

Recent technical advances in mass spectrometry (MS) have propelled this technology to the forefront of methods employed in metabolome analysis. Here, we compare two distinct analytical approaches based on MS for their potential in revealing specific metabolic footprints of yeast single-deletion mutants. Filtered fermentation broth samples were analyzed by GC-MS and direct infusion ESI-MS. The potential of both methods in producing specific and, therefore, discriminant metabolite profiles was evaluated using samples from several yeast deletion mutants grown in batch-culture conditions with glucose as the carbon source. The mutants evaluated were cat8, gln3, ino2, opi1, and nil1, all with deletion of genes involved in nutrient sensing and regulation. From the analysis, we found that both methods can be used to classify mutants, but the classification depends on which metabolites are measured. Thus, the GC-MS method is good for classification of mutants with altered nitrogen regulation as it primarily measures amino acids, whereas this method cannot classify mutants involved in regulation of phospholipids metabolism as well as the direct infusion MS (DI-MS) method. From the analysis, we find that it is possible to discriminate the mutants in both the exponential and stationary growth phase, but the data from the exponential growth phase provide more physiological relevant information. Based on the data, we identified metabolites that are primarily involved in discrimination of the different mutants, and hereby providing a link between high-throughput metabolome analysis, strain classification, and physiology.