21 research outputs found
Physicochemical parameters affecting the electrospray ionization efficiency of amino acids after acylation
Electrospray
ionization (ESI) is widely used in liquid chromatography
coupled to mass spectrometry (LC–MS) for the analysis of biomolecules.
However, the ESI process is still not completely understood, and it
is often a matter of trial and error to enhance ESI efficiency and,
hence, the response of a given set of compounds. In this work we performed
a systematic study of the ESI response of 14 amino acids that were
acylated with organic acid anhydrides of increasing chain length and
with poly(ethylene glycol) (PEG) changing certain physicochemical
properties in a predictable manner. By comparing the ESI response
of 70 derivatives, we found that there was a strong correlation between
the calculated molecular volume and the ESI response, while correlation
with hydrophobicity (log <i>P</i> values), p<i>K</i><sub>a</sub>, and the inverse calculated surface tension was significantly
lower although still present, especially for individual derivatized
amino acids with increasing acyl chain lengths. Acylation with PEG
containing five ethylene glycol units led to the largest gain in ESI
response. This response was maximal independent of the calculated
physicochemical properties or the type of amino acid. Since no actual
physicochemical data is available for most derivatized compounds,
the responses were also used as input for a quantitative structure–property
relationship (QSPR) model to find the best physicochemical descriptors
relating to the ESI response from molecular structures using the amino
acids and their derivatives as a reference set. A topological descriptor
related to molecular size (SPAN) was isolated next to a descriptor
related to the atomic composition and structural groups (BIC0). The
validity of the model was checked with a test set of 43 additional
compounds that were unrelated to amino acids. While prediction was
generally good (<i>R</i><sup>2</sup> > 0.9), compounds
containing
halogen atoms or nitro groups gave a lower predicted ESI response
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
Development of immunopurification and capillary electrophoresis methodologies for the study of glycoproteins as potential cancer biomarkers
Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Análisis Instrumental y Química Ambiental. Fecha de lectura: 20-07-201
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
Statistical evaluation of CZE-UV and CZE-ESI-MS data of intact α-1-acid glycoprotein isoforms for their use as potential biomarkers in bladder cancer
α-1-acid glycoprotein (AGP) is a highly heterogeneous protein that presents a vast number of isoforms (molecules of the protein differing in its peptidic and/or glycosidic moieties). In the last years, several authors have studied the potential use of AGP as a cancer biomarker. These studies focus on the correlation of different features of AGP structure (i.e. fucosylation, antennarity) with cancer or on the total protein blood concentration. In this study, the potential of CZE-UV and CZE-ESI-MS analysis of intact AGP isoforms to study the correlation of this protein with bladder cancer is shown. Samples from 16 individuals (eight healthy, eight bladder cancer) were analyzed and characterized in great detail including data on intact protein isoforms and on released glycans. The analytical data were evaluated employing different statistical techniques (ANOVA; principal component analysis, PCA; linear discriminant analysis; and partial least squares-discriminant analysis). Statistical differences between the two groups of study were observed. The best results were obtained by linear discriminant analysis of the CZE-ESI-MS data for intact AGP isoforms (93.75% of correct classification). Due to MS characterization, it can be observed that differences between the samples are mainly due to higher abundance of AGP isoforms containing tri- and tetra-antennary fucosylated oligosaccharides in cancer patients. The results show the great potential of CE-MS in combination with advanced data processing for the use of intact protein isoforms as disease biomarkers.Peer reviewe
Statistical evaluation of CZE-UV and CZE-ESI-MS data of intact alpha-1-acid glycoprotein isoforms for their use as potential biomarkers
Peer reviewe
Evaluation of the effect of the immunopurification-based procedures on the CZE-UV and CZE-ESI-TOF-MS determination of isoforms of intact alpha-1-acid glycoprotein from human serum
Differences in a-1-acid glycoprotein (AGP) peptidic and glycan moieties originate several
isoforms, whose modifications have been related to different pathophysiological situa-
tions. 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 puri-
fication 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 deter-
mination 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.Peer reviewe
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. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.Peer Reviewe
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 car-
diovascular 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 capil-
lary 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 interfer-
ents 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 chro-
matography (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.Sara Ongay and Izaskun Lacunza acknowledge CSIC for contracts
to perform Ph.D. Theses. Drs. Enrique Caso and Maria del Val Toledo
are acknowledged for providing the serum samples. Financial sup-
port from the Spanish Ministry of Science and Innovation (projects
CTQ 2006-05214, HH 2006-0013, and PSE-010000-2008-6) and
Comunidad de Madrid (project S-GEN-0247-2006) is acknowledged.Peer reviewe