Rico Derks

Fc specific IgG glycosylation profiling by robust nano-reverse phase HPLC-MS using a sheath-flow ESI sprayer interface

Biological activities of immunoglobulin G such as effector functions via Fc receptor interactions are influenced by Fc-linked N-glycans. Here we describe a fast, robust and sensitive nano-LC-ESI-MS method for detailed subclass specific analysis of IgG Fc N-glycosylation. A sheath-flow ESI sprayer was used as a sensitive zero dead volume plug-and-play interface for online MS coupling, generating a very constant spray and ionization over the entire LC gradient. The propionic acid containing sheath-liquid effectively suppressed TFA gas-phase ion-pairing, enabling the use of TFA containing mobile phases. The fixed position of the sheath-flow ESI sprayer, far away from the glass capillary inlet, reduced MS contamination as compared to conventional nano-ESI. The method was found to be suitable for fast and detailed subclass specific IgG Fc N-glycosylation profiling in human plasma. The obtained subclass specific IgG Fc N-glycosylation profiles were processed automatically using in house developed software tools. For each of the IgG subclasses the 8 major glycoforms showed an interday analytical variation below 5%. The method was used to profile the IgG Fc N-glycosylation of 26 women at several time points during pregnancy and after delivery, revealing pregnancy-associated changes in IgG galactosylation, sialylation and incidence of bisecting N-acetylglucosamine.

Ultra-low flow electrospray ionization-mass spectrometry for improved ionization efficiency in phosphoproteomics.

The potential benefits of ultra-low flow electrospray ionization (ESI) for the analysis of phosphopeptides in proteomics was investigated. First, the relative flow dependent ionization efficiency of nonphosphorylated vs multiplyphosphorylated peptides was characterized by infusion of a five synthetic peptide mix with zero to four phophorylation sites at flow rates ranging from 4.5 to 500 nL/min. Most importantly, similar to what was found earlier by Schmidt et al., it has been verified that at flow rates below 20 nL/min the relative peak intensities for the various peptides show a trend toward an equimolar response, which would be highly beneficial in phosphoproteomic analysis. As the technology to achieve liquid chromatography separation at flow rates below 20 nL/min is not readily available, a sheathless capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) strategy based on the use of a neutrally coated separation capillary was used to develop an analytical strategy at flow rates as low as 6.6 nL/min. An in-line preconcentration technique, namely, transient isotachophoresis (t-ITP), to achieve efficient separation while using larger volume injections (37% of capillary thus 250 nL) was incorporated to achieve even greater sample concentration sensitivities. The developed t-ITP-ESI-MS strategy was then used in a direct comparison with nano-LC-MS for the detection of phosphopeptides. The comparison showed significantly improved phosphopeptide sensitivity in equal sample load and equal sample concentration conditions for CE-MS while providing complementary data to LC-MS, demonstrating the potential of ultra-low flow ESI for the analysis of phosphopeptides in liquid based separation techniques.

Capillary electrophoresis-time of flight-mass spectrometry using noncovalently bilayer-coated capillaries for the analysis of amino acids in human urine

A capillary electrophoresis‐time of flight‐mass spectrometry (CE‐TOF‐MS) method for the analysis of amino acids in human urine was developed. Capillaries noncovalently coated with a bilayer of Polybrene (PB) and poly(vinyl sulfonate) (PVS) provided a considerable EOF at low pH, thus facilitating the fast separation of amino acids using a BGE of 1 M formic acid (pH 1.8). The PB–PVS coating proved to be very consistent yielding stable CE‐MS patterns of amino acids in urine with favorable migration time repeatability (RSDs <2%). The relatively low sample loading capacity of CE was circumvented by an in‐capillary preconcentration step based on pH‐mediated stacking allowing 100‐nL sample injection (i.e. ca. 4% of capillary volume). As a result, LODs for amino acids were down to 20 nM while achieving satisfactory separation efficiencies. Preliminary validation of the method with urine samples showed good linear responses for the amino acids (R2 >0.99), and RSDs for peak areas were <10%. Special attention was paid to the influence of matrix effects on the quantification of amino acids. The magnitude of ion suppression by the matrix was similar for different urine samples. The CE‐TOF‐MS method was used for the analysis of urine samples of patients with urinary tract infection (UTI). Concentrations of a subset of amino acids were determined and compared with concentrations in urine of healthy controls. Furthermore, partial least squares–discriminant analysis (PLS–DA) of the CE‐TOF‐MS dataset in the 50–450 m/z region showed a distinctive grouping of the UTI samples and the control samples. Examination of score and loadings plot revealed a number of compounds, including phenylalanine, to be responsible for grouping of the samples. Thus, the CE‐TOF‐MS method shows good potential for the screening of body fluids based on the analysis of endogenous low‐molecular weight metabolites such as amino acids and related compounds.

Alignment of capillary electrophoresis–mass spectrometry datasets using accurate mass information

Capillary electrophoresis–mass spectrometry (CE–MS) is a powerful technique for the analysis of small soluble compounds in biological fluids. A major drawback of CE is the poor migration time reproducibility, which makes it difficult to combine data from different experiments and correctly assign compounds. A number of alignment algorithms have been developed but not all of them can cope with large and irregular time shifts between CE–MS runs. Here we present a genetic algorithm designed for alignment of CE–MS data using accurate mass information. The utility of the algorithm was demonstrated on real data, and the results were compared with one of the existing packages. The new algorithm showed a significant reduction of elution time variation in the aligned datasets. The importance of mass accuracy for the performance of the algorithm was also demonstrated by comparing alignments of datasets from a standard time-of-flight (TOF) instrument with those from the new ultrahigh resolution TOF maXis (Bruker Daltonics).

CE-MS for metabolic profiling of volume-limited urine samples: application to accelerated aging TTD mice.

Metabolic profiling of biological samples is increasingly used to obtain more insight into the pathophysiology of diseases. For translational studies, biological samples from animal models are explored; however, the volume of these samples can be a limiting factor for metabolic profiling studies. For instance, only a few microliters of urine is often available from small animals like mice. Hence, there is a need for a tailor-made analytical method for metabolic profiling of volume-limited samples. In the present study, the feasibility of capillary electrophoresis time-of-flight mass spectrometry (CE-ToF-MS) for metabolic profiling of urine from mice is evaluated. Special attention is paid to the analytical workflow; that is, such aspects as sample preparation, analysis, and data treatment are discussed from the metabolomics viewpoint. We show that metabolites belonging to several chemical families can be analyzed in mouse urine with the CE-ToF-MS method using minimal sample pretreatment and an in-capillary preconcentration procedure. This exemplifies the advantages of CE-ToF-MS for metabolic profiling of volume-limited samples as loss of material is minimized. The feasibility of the CE-ToF-MS-based workflow for metabolic profiling is illustrated by the analysis of urine samples from wild-type as well as from TTD mutant mice, which are a model for the accelerated aging, with osteoporosis being one of the main hallmarks.

Explorative analysis of urine by capillary electrophoresis-mass spectrometry in chronic patients with complex regional pain syndrome.

Complex Regional Pain Syndrome (CRPS) is characterized by various combinations of sensory, autonomic and motor disturbances. Pain disproportionate to the severity and duration of the inciting event is the most devastating symptom. Diagnosis of CRPS is difficult as the underlying mechanisms remain unclear. To try to derive metabolic indicators potentially characteristic for CRPS, we applied capillary electrophoresis time-of-flight mass spectrometry (CE-ToF-MS) to the explorative analysis of urine. The CE-ToF-MS method provided fast and stable metabolic profiles of urine samples. The mean intraday and interday CVs were <2% and <9% for migration times and peak areas, respectively, demonstrating robustness of the method. With the use of multivariate chemometric analysis, discrimination between urine samples from CRPS patients and controls was obtained, emphasizing differences in metabolic signatures between CRPS-diseased patients and controls. Several compounds, such as 3-methylhistidine, were responsible for discriminating the samples. The biological relevance of these compounds with regard to CRPS is discussed. Thus, CE-ToF-MS-based metabolic profiling of urine from CRPS patients and controls revealed metabolites that differentiate between diseased and control, illustrating the usefulness of this approach to get more insight into the pathology underlying CRPS.

Analytical pitfalls and challenges in clinical metabolomics

Metabolomics-based strategies have become an integral part of modern clinical research, allowing for a better understanding of pathophysiological conditions and disease mechanisms, as well as providing innovative tools for more adequate diagnostic and prognosis approaches. Metabolomics is considered an essential tool in precision medicine, which aims for personalized prevention and tailor-made treatments. Nevertheless, multiple pitfalls may be encountered in clinical metabolomics during the entire workflow, hampering the quality of the data and, thus, the biological interpretation. This review describes the challenges underlying metabolomics-based experiments, discussing step by step the potential pitfalls of the analytical process, including study design, sample collection, storage, as well as preparation, chromatographic and electrophoretic separation, detection and data analysis. Moreover, it offers practical solutions and strategies to tackle these challenges, ensuring the generation of high-quality data.

Ultra high performance liquid chromatography-time of flight mass spectrometry for analysis of avocado fruit metabolites: Method evaluation and applicability to the analysis of ripening degrees

We have developed an analytical method using UHPLC-UV/ESI-TOF MS for the comprehensive profiling of the metabolites found in the methanolic extracts of 13 different varieties of avocado at two different ripening degrees. Both chromatographic and detection parameters were optimized in order to maximize the number of compounds detected and the sensitivity. After achieving the optimum conditions, we performed a complete analytical validation of the method with respect to its linearity, sensitivity, precision, accuracy and possible matrix effects. The LODs ranged from 1.64 to 730.54 ppb (in negative polarity) for benzoic acid and chrysin, respectively, whilst they were found within the range from 0.51 to 310.23 ppb in positive polarity. The RSDs for repeatability test did not exceed 7.01% and the accuracy ranged from 97.2% to 102.0%. Our method was then applied to the analysis of real avocado samples and advanced data processing and multivariate statistical analysis (PCA, PLS-DA) were carried out to discriminate/classify the examined avocado varieties. About 200 compounds belonging to various structural classes were tentatively identified; we are certain about the identity of around 60 compounds, 20 of which have been quantified in terms of their own commercially available standard.

Coupling porous sheathless interface MS with transient-ITP in neutral capillaries for improved sensitivity in glycopeptide analysis

IgG antibodies are modulated in their function by the specific structure of the N‐glycans attached to their Fc (fragment crystallizable) portions. However, the glycosylation analysis of antigen‐specific IgGs is a challenging task as antibody levels to a given antigen only represent a fraction of the total IgG levels. Here, we investigated the use of a transient‐ITP (t‐ITP)—MS method for highly sensitive IgG1 glycosylation profiling as a complementary method to a high‐throughput nano‐RPLC‐MS method. It was found that t‐ITP‐CZE using neutrally coated separation capillaries with a large volume injection (37% of capillary volume) and interfaced to MS with a sheathless porous sprayer yielded a 40‐fold increase in sensitivity for IgG1 Fc glycopeptide analysis when compared to the conventional strategy. Furthermore, the glycoform profiles found with the t‐ITP‐CZE strategy were comparable to those from nano‐RPLC‐MS. In conclusion, the use of the highly sensitive t‐ITP‐CZE‐MS method will provide information on IgG Fc glycosylation for those samples with IgG1 concentrations below the LODs of the conventional method.

Derivatization of the tricarboxylic acid cycle intermediates and analysis by online solid-phase extraction–liquid chromatography–mass spectrometry with positive-ion electrospray ionization

The analysis of cellular metabolic processes is of fundamental biological interest. Cellular metabolites, such as the intermediates of the tricarboxylic acid (TCA) cycle, provide essential information about the metabolic state of the cell. Not only is the TCA cycle a key factor in the energy regulation within aerobic cells, it possibly also plays a role in cell signaling. This paper describes a novel derivatization strategy, using the empirically selected N-methyl-2-phenylethanamine as derivatization reagent with a carbodiimide as co-reagent, for the selective derivatization of carboxylic acids, such as the di- and tri-carboxylic acids of the TCA cycle. Optimization of the derivatization protocol is described. This procedure enables analysis of the derivatives using on-line solid-phase extraction and reversed-phase liquid chromatography in combination with sensitive positive-ion electrospray ionization mass spectrometry. The complete procedure, involving the use of core-shell silica column material, allows the rapid analysis of TCA cycle intermediates in sample matrices, here shown for pig heart tissue extracts, with a good linearity over 3-4 orders of magnitude. Detection limits range from 12 to 1000 nM, depending on the analyte.