Sara Ongay

Glycopeptide enrichment and separation for protein glycosylation analysis

Protein glycosylation plays key roles in many biological processes. In addition, alterations in protein glycosylation have been related to different diseases, as well as may affect the properties of recombinant proteins used as human therapeutics. For this reason, protein glycosylation analysis is of main interest in biomedical and biopharmaceutical research. Although recent advances in LC-MS analysis have made possible glycoprotein glycosylation site identification, characterization of glycoprotein glycan structures, as well as glycoprotein identification and quantification, protein glycosylation analysis in complex samples still remains a difficult task. This is due to low proportions of glycopeptides in comparison to peptides obtained after glycoprotein digestion, the suppression of the glycopeptide MS signals in the presence of peptides, and the high heterogeneity of glycopeptides. Thus, in the recent years, continuous efforts have been devoted to the development of glycopeptide enrichment and separation strategies to facilitate and improve glycoprotein glycosylation analysis in complex samples. This review summarizes the different methodologies that can be employed for glycopeptide enrichment/separation from complex samples including methods based on lectin affinity enrichment, covalent interactions, or chromatographic separations and solid-phase extraction.

Quantification of free and total desmosine and isodesmosine in human urine by liquid chromatography tandem mass spectrometry: A comparison of the surrogate-analyte and the surrogate-matrix approach for quantitation

In spite of the data suggesting the potential of urinary desmosine (DES) and isodesmosine (IDS) as biomarkers for elevated lung elastic fiber turnover, further validation in large-scale studies of COPD populations, as well as the analysis of longitudinal samples is required. Validated analytical methods that allow the accurate and precise quantification of DES and IDS in human urine are mandatory in order to properly evaluate the outcome of such clinical studies. In this work, we present the development and full validation of two methods that allow DES and IDS measurement in human urine, one for the free and one for the total (free+peptide-bound) forms. To this end we compared the two principle approaches that are used for the absolute quantification of endogenous compounds in biological samples, analysis against calibrators containing authentic analyte in surrogate matrix or containing surrogate analyte in authentic matrix. The validated methods were employed for the analysis of a small set of samples including healthy never-smokers, healthy current-smokers and COPD patients. This is the first time that the analysis of urinary free DES, free IDS, total DES, and total IDS has been fully validated and that the surrogate analyte approach has been evaluated for their quantification in biological samples. Results indicate that the presented methods have the necessary quality and level of validation to assess the potential of urinary DES and IDS levels as biomarkers for the progression of COPD and the effect of therapeutic interventions.

Evaluation of the effect of the immunopurification-based procedures on the CZE-UV and CZE-ESI-TOF-MS determination of isoforms of intact α-1-acid glycoprotein from human serum

Differences in α‐1‐acid glycoprotein (AGP) peptidic and glycan moieties originate several isoforms, whose modifications have been related to different pathophysiological situations. Differences in the isoforms of AGP existing in serum of individuals suffering from different diseases compared to healthy ones could be potentially used as biomarkers. CZE has been proven to be a useful technique for the analysis of glycoprotein isoforms. However, direct CZE analysis of AGP isoforms in serum samples needs efficient purification methods that allow the protein analysis. In this work two new and fast methods to purify AGP from human serum are evaluated in regard to their effect on the determination of isoforms of the intact glycoprotein by CZE‐UV and by a developed CZE‐ESI‐TOF‐MS method. Both preparation methods, which differ in the pre‐treatment of the sample prior to an anti‐AGP immunochromatographic step are shown to be adequate to analyze isoforms of intact AGP. Comparison of both purification methods by CZE‐UV and CZE‐ESI‐TOF‐MS indicates that serum AGP purified without acidic precipitation as pre‐treatment is more adequate due to AGP higher yield, which leads to better CZE‐Mass spectra. Both CZE methods show no indication that acidic precipitation influences the glycosylation (including sialylation) of AGP.

Development of a fast and simple immunochromatographic method to purify alpha 1-acid glycoprotein from serum for analysis of its isoforms by capillary electrophoresis

Alpha 1-acid glycoprotein (AGP) is a very heterogeneous glycoprotein presenting several isoforms due to variations in its polypeptidic and glycosidic moieties. Differences in AGP isoforms between healthy and diseased individuals have been related to different pathological situations such as cancer or cardiovascular diseases, among others. Capillary electrophoresis study of the role of AGP isoforms as biomarkers requires prior purification of AGP from biological samples. Current AGP purification methods are time- and labour-consuming, and generally they have not been proven to be compatible with capillary electrophoresis analysis. In this work, different methods for AGP purification from human serum are developed and compared. The applicability of acidic precipitation and immunoaffinity chromatographic methods for AGP purification are studied. Two different immunoaffinity approaches are employed; in the first one, interferents present in the AGP sample are captured and removed, and in the second one, AGP is retained in a house-made anti-AGP column, being in this way isolated from the rest of interferents of the sample. Best results in AGP purification from human serum to be analyzed by capillary zone electrophoresis (CZE) were obtained when acidic purification was combined with immunoaffinity chromatography (IAC) employing the house-made anti-AGP column. The method was shown not to alter the proportion of AGP peaks due to isoforms existing in AGP samples. The applicability of this fast and easy purification method developed for analyzing by CZE isoforms of AGP from natural serum samples by CZE is demonstrated.

Development of CE methods to analyze potential components of the angiogenic glycoprotein vascular endothelial growth factor 165

The vascular endothelial growth factor 165 (VEGF165) is the predominant form of the complex VEGF family. This glycoprotein has, among others, an angiogenic effect in many physiological and pathological events. For this reason, its roles as a biomarker and as a therapeutic drug have been considered. However, very little is known about the existence of different forms of VEGF165 arising from glycosylation and other potential PTMs. This aspect is crucial because it is known that for other glycoproteins the ratio between these isoforms actually acts as a biomarker for certain diseases and other physiological states. In addition, for therapeutic use of glycoproteins it is known that the biological activity may differ for the various isoforms. In this work CE methods to separate up to seven peaks without baseline resolution containing various forms of VEGF165 are developed. Using a computer program previously developed in‐house peak assignment could be performed with accuracy close to 100%. In this way, comparison between recombinant human VEGF165 expressed in insect cells, which is a glycosylating system, and in Escherichia coli cells, which are unable of performing glycosylation of proteins, has been possible. The methods developed, besides providing information about the existence of several forms of VEGF165, mean a starting point that permits the study of the role of VEGF165 as a potential biomarker of different diseases and physiological processes and to perform quality control of the recombinant drug during manufacturing. To the best of our knowledge this is the first time that CE methods for VEGF165 have been developed.

CIEF and MALDI-TOF-MS methods for analyzing forms of the glycoprotein VEGF165

The vascular endothelial growth factor (VEGF) is involved in different sicknesses (cardiovascular diseases, cancer, and other). Out of the many components of the VEGF family, the A splice variant with 165 amino acids (VEGF165) is the main component. In spite of the potential as biomarker that this protein has, information about its physico‐chemical characteristics is scarce. In this study CIEF and MALDI‐TOF‐MS methods for intact recombinant human VEGF165 are developed and applied to analyze this glycoprotein expressed in glycosylating (Sf 21 insect cells) and non‐glycosylating (Escherichia coli) systems. Different parameters influencing the CIEF separation were studied. The developed CIEF method allowed for the separation of up to seven peaks in the VEGF165 expressed in insect cells and up to three in VEGF165 expressed in E. coli. The use of the presented method permits the estimation of the apparent pI of the different forms of VEGF165 expressed in insect cells to be in a range of 6.8–8.2. The three peaks with intermediate pI values are observed in the protein expressed in both systems, insect cells and E. coli. The MALDI‐TOF‐MS method enabled to a rapid partial characterization of VEGF165 based on its MS fingerprint. MALDI‐MS analysis of VEGF165 expressed in insect cells shows the presence of, at least, four forms or groups of forms of VEGF165 as a result of the different PTMs of the protein. According to the MALDI‐MS analysis, VEGF165 expressed in E. coli was produced as a very homogeneous protein, although the results suggest the existence of some PTMs in the protein. The patterns of VEGF165 of both origins obtained by CIEF and MALDI‐MS indicate the possibility of using these analytical methods to compare samples from people with different pathophysiological conditions. This work is thus a starting point to make possible the study of the role of the various forms of VEGF165 as biomarkers. Finally, to the best of our knowledge, this is the first time that intact VEGF165 has been analyzed by CIEF and MALDI‐TOF‐MS.

Immunoaffinity, Capillary Electrophoresis, and Statistics for Studying Intact Alpha 1-Acid Glycoprotein Isoforms as an Atherothrombosis Biomarker

Variations in the amino acid sequence, glycosylation, and/or other posttranslational modifications in glycoproteins give rise to different molecules of the glycoprotein called forms. Qualitative and/or quantitative alterations in these forms are related to pathophysiological situations in the individuals. In this study, a methodology to analyze these differences in forms of the alpha 1-acid glycoprotein (AGP) between healthy individuals and patients with two different vascular diseases is detailed. The whole methodology includes a sample preparation method based on immunochromatography, a capillary electrophoresis method for separation of AGP peaks (isoforms), and statistical methods (Linear Discriminant Analysis) for sample classification. As a result, it is shown that the methodology proposed allows studying the role of AGP isoforms as potential vascular disease biomarkers.

Electron Transfer and Collision Induced Dissociation of Non-Derivatized and Derivatized Desmosine and Isodesmosine

AbstractElectron transfer dissociation (ETD) has attracted increasing interest due to its complementarity to collision-induced dissociation (CID). ETD allows the direct localization of labile post-translational modifications, which is of main interest in proteomics where differences and similarities between ETD and CID have been widely studied. However, due to the fact that ETD requires precursor ions to carry at least two charges, little is known about differences in ETD and CID of small molecules such as metabolites. In this work, ETD and CID of desmosine (DES) and isodesmosine (IDS), two isomers that due to the presence of a pyridinium group can carry two charges after protonation, are studied and compared. In addition, the influence of DES/IDS derivatization with propionic anhydride and polyethyleneglycol (PEG) reagents on ETD and CID was studied, since this is a common strategy to increase sensitivity and to facilitate the analysis by reversed-phase chromatography. Clear differences between ETD and CID of non-derivatized and derivatized-DES/IDS were observed. While CID is mainly attributable to charge-directed fragmentation, ETD is initiated by the generation of a hydrogen atom at the initial protonation site and its subsequent transfer to the pyridinium ring of DES/IDS. These differences are reflected in the generation of complex CID spectra dominated by the loss of small, noninformative molecules (NH3, CO, H2O), while ETD spectra are simpler and dominated by characteristic side-chain losses. This constitutes a potential advantage of ETD in comparison to CID when employed for the targeted analysis of DES/IDS in biological samples. FigureA mechanistic study of electron transfer dissociation (ETD) and collision-induced dissociation (CID) of labeled and free desmosine and isodesmosine provides evidence that CID is mainly due to charge-directed fragmentation while ETD is initiated by the generation of a hydrogen atom at the initial protonation site, and its subsequent transfer to the pyridinium ring.

Assessment of Sample Preparation Bias in Mass Spectrometry-Based Proteomics

For mass spectrometry-based proteomics, the selected sample preparation strategy is a key determinant for information that will be obtained. However, the corresponding selection is often not based on a fit-for-purpose evaluation. Here we report a comparison of in-gel (IGD), in-solution (ISD), on-filter (OFD), and on-pellet digestion (OPD) workflows on the basis of targeted (QconCAT-multiple reaction monitoring (MRM) method for mitochondrial proteins) and discovery proteomics (data-dependent acquisition, DDA) analyses using three different human head and neck tissues (i.e., nasal polyps, parotid gland, and palatine tonsils). Our study reveals differences between the sample preparation methods, for example, with respect to protein and peptide losses, quantification variability, protocol-induced methionine oxidation, and asparagine/glutamine deamidation as well as identification of cysteine-containing peptides. However, none of the methods performed best for all types of tissues, which argues against the existence of a universal sample preparation method for proteome analysis.

Prioritization of COPD protein biomarkers, based on a systematic study of the literature

Chronic Obstructive Pulmonary Disease (COPD) is a chronic lung disease mostly due to smoking and until now diagnosed by spirometry (post bronchodilator FEV1/FVC <70%). However, in spite of the usefulness of FEV1 as diagnostic and prognostic tool, it has proven to be a weak indicator of future exacerbations, unable to predict lung function decline within COPD patients, as well as unable to identify the smokers “susceptible” to developing COPD at an early stage. Thus, there is an urgent need for biomarkers that address these questions and support clinical decision making in the diagnosis and treatment of (early) COPD. In this respect, considerable efforts have been devoted to identifying protein biomarkers that enable a better understanding of this complex disease and leading to better diagnostic and prognostic tools. However, in spite of the wide range of candidates that have been suggested as potentially useful COPD biomarkers, most remained at the level of the initial discovery, and only fibrinogen has been approved by the Food and Drug Administration (FDA) as predictor for all-cause mortality and COPD exacerbations. There is thus a need for future investigations of these biomarkers in large-scale and well characterized studies in order to prove their usefulness as surrogate endpoints. Based on this, the aim of the present review is to advance COPD biomarker development by providing a comprehensive overview of protein biomarker candidates which have been evaluated in clinical studies and prioritize them according to their potential of becoming valid, clinically useful COPD biomarkers.