A Proteomics Data Mining Strategy for the Identification of Quinoa Grain Proteins with Potential Immunonutritional Bioactivities.

Quinoa proteins are attracting global interest for their wide amino acid profile and as a promising source for the development of biomedical treatments, including those against immune-mediated diseases. However, information about the bioactivity of quinoa proteins is scarce. In this study, a quinoa grain proteome map obtained by label-free mass spectrometry-based shotgun proteomics was investigated for the identification of quinoa grain proteins with potential immunonutritional bioactivities, including those related to cancer. After carefully examining the sequence similarities of the 1211 identified quinoa grain proteins against already described bioactive proteins from other plant organisms, 71, 48, and 3 of them were classified as antimicrobial peptides (AMPs), oxidative stress induced peptides (OSIPs), and serine-type protease inhibitors (STPIs), respectively, suggesting their potential as immunomodulatory, anti-inflammatory, and anticancer agents. In addition, data interpretation using Venn diagrams, heat maps, and scatterplots revealed proteome similarities and differences with respect to the AMPs, OSIPs, and STPIs, and the most relevant bioactive proteins in the predominant commercial quinoa grains (i.e., black, red, white (from Peru), and royal (white from Bolivia)). The presented proteomics data mining strategy allows easy screening for potentially relevant quinoa grain proteins and commercial classes for immunonutrition, as a basis for future bioactivity testing.

On-line Immobilized Enzyme Microreactor Capillary Zone Electrophoresis-Mass Spectrometry for Peptide Mapping.

Peptide mapping is a routine procedure for protein characterization in proteomics. This bottom-up analysis requires digestion of proteins into peptides before liquid chromatography- or capillary zone electrophoresis-mass spectrometry (LC-MS or CZE-MS, respectively). Proteins are usually digested off-line using proteolytic enzymes, typically trypsin, in solution or immobilized on appropriate supports. As an alternative, here we describe on-line immobilized enzyme microreactor capillary zone electrophoresis-mass spectrometry (IMER-CZE-MS) for a straightforward, rapid, and efficient protein digestion followed by separation, detection, and characterization of the generated peptides.

Site-Specific N-Linked Glycosylation Analysis of Human Carcinoembryonic Antigen by Sheathless Capillary Electrophoresis-Tandem Mass Spectrometry.

With 28 potential N-glycosylation sites, human carcinoembryonic antigen (CEA) bears an extreme amount of N-linked glycosylation, and approximately 60% of its molecular mass can be attributed to its carbohydrates. CEA is often overexpressed and released by many solid tumors, including colorectal carcinomas. CEA displays an impressive heterogeneity and variability in sugar content; however, site-specific distribution of carbohydrate structures has not been reported so far. The present study investigated CEA samples purified from human colon carcinoma and human liver metastases and enabled the characterization of 21 out of 28 potential N-glycosylation sites with respect to their occupancy. The coverage was achieved by a multienzymatic digestion approach with specific enzymes, such as trypsin, endoproteinase Glu-C, and the nonspecific enzyme, Pronase, followed by analysis using sheathless CE-MS/MS. In total, 893 different N-glycopeptides and 128 unique N-glycan compositions were identified. Overall, a great heterogeneity was found both within (micro) and in between (macro) individual N-glycosylation sites. Moreover, notable differences were found on certain N-glycosylation sites between primary adenocarcinoma and metastatic tumor in regard to branching, bisection, sialylation, and fucosylation. Those features, if further investigated in a targeted manner, may pave the way toward improved diagnostics and monitoring of colorectal cancer progression and recurrence. Raw mass spectrometric data and Skyline processed data files that support the findings of this study are available in the MassIVE repository with the identifier MSV000086774 [DOI: 10.25345/C5Z50X].

On-Line Immunoaffinity Solid-Phase Extraction Capillary Electrophoresis-Mass Spectrometry for the Analysis of Serum Transthyretin.

The analysis of low abundant proteins in biological fluids by capillary electrophoresis (CE) is particularly problematic due to the typically poor concentration limits of detection of microscale separation techniques. Another important issue is sample matrix complexity that requires an appropriate cleanup. Here, we describe an on-line immunoaffinity solid-phase extraction capillary electrophoresis-mass spectrometry (IA-SPE-CE-MS) method for the immunoextraction, preconcentration, separation, detection, and characterization of serum transthyretin (TTR). TTR is a protein biomarker related to diverse types of amyloidosis, such as familial amyloidotic polyneuropathy type I (FAP-I), which is the most common hereditary systemic amyloidosis.

Multivariate data analysis for the detection of human alpha-acid glycoprotein aberrant glycosylation in pancreatic ductal adenocarcinoma.

Relative quantification of human alpha-acid glycoprotein (hAGP) glycan isomers using [12C6]/[13C6]-aniline in combination with multivariate data analysis is proposed as an efficient method for the identification of pancreatic ductal adenocarcinoma (PDAC) glycan biomarkers in serum samples. Intact and desialylated glycans from hAGP, purified from serum samples of patients with PDAC and chronic pancreatitis (ChrP), were labeled with aniline and analyzed by µZIC-HILIC-MS. Afterwards, partial least squares discriminant analysis (PLS-DA) was applied to the relative areas obtained for all glycan isomers in the different samples: pathological (ChrP or PDAC) versus healthy samples. Seven intact glycan isomers with α2-6 linked sialic acids, five of them also fucosylated, were the most meaningful to distinguish between PDAC and ChrP patients. The desialylated glycan isomers also identified by PLS-DA as potential biomarker candidates confirmed that antenna but also core fucosylation could be involved in PDAC. The analysis of intact and desialylated glycan isomers in combination with the multivariate data analysis revealed that the triantennary glycan with two fucoses of hAGP could have in the future a relevant role in the differentiation of patients with PDAC from those with ChrP. SIGNIFICANCE: Multivariate data analysis is currently being used in many omics fields for biomarker discovery. However, to date, no glycomics studies have applied chemometric tools combined with mass spectrometry in a preclinical research. In this work, this methodology has been used to identify altered glycosylation of human alpha-acid glycoprotein in pancreatic ductal adenocarcinoma (PDAC). The obtained results reveal that the triantennary glycan with two fucoses could have a great biomarker potential as it was relevant to differentiate PDAC and chronic pancreatitis (ChrP) patients.

Identification of antihypertensive peptides in nutraceuticals by capillary electrophoresis-mass spectrometry.

We present capillary electrophoresis-mass spectrometry (CE-MS) in combination with advanced chemometric tools for the analysis of bioactive compounds in food, in particular for the identification of antihypertensive peptides in a nutraceutical derived from a bovine milk protein hydrolysate. Different extracts of the nutraceutical were analyzed by CE-MS, and the electropherograms were processed using a novel data analysis workflow that included regions of interest (ROIs) compression and multivariate curve resolution alternating least squares (MCR-ALS). MCR-ALS permitted the description of the nutraceutical extract as ten characteristic components with their electrophoretic profiles and mass spectra. Twenty-two compounds were tentatively identified as antihypertensive bovine casein fragments through a mass search in a database of bioactive peptides. The identity of 17 antihypertensive peptides was reliably confirmed by capillary electrophoresis-tandem mass spectrometry. The proposed analytical approach demonstrated the potential to obtain a characteristic and activity-related fingerprint for quality control and authentication of the antihypertensive nutraceutical.

Analysis of Circulating microRNAs and Their Post-Transcriptional Modifications in Cancer Serum by On-Line Solid-Phase Extraction-Capillary Electrophoresis-Mass Spectrometry.

In this paper, an on-line solid-phase extraction capillary electrophoresis-mass spectrometry (SPE-CE-MS) method is described for the purification, preconcentration, separation, and characterization of endogenous microRNA (miRNA) and their post-transcriptional modifications in serum. First, analysis by CE-MS was optimized using a standard mixture of hsa-miR-21-5p (miR-21-5p) and hsa-let-7g-5p (let-7g-5p). For SPE-CE-MS, a commercial silicon carbide (SiC) resin was used to prepare the microcartridges. Under the optimized conditions with standards, the microcartridge lifetime (>25 analyses) and repeatability (2.8% RSD for the migration times; 4.4 and 6.4% RSD for the miR-21-5p and let-7g-5p peak areas, respectively) were good, the method was linear between 25 and 100 nmol·L-1, and the limit of detection (LOD) was around 10 nmol·L-1 (50 times lower than by CE-MS). In order to analyze human serum samples, an off-line sample pretreatment based on phenol/chloroform/isoamyl alcohol (PCA) extraction was necessary prior to SPE-CE-MS. The potential of the SPE-CE-MS method to screen for B-cell chronic lymphocytic leukemia (CLL) was demonstrated by an analysis of serum samples from healthy controls and patients. MicroRNAs, specifically miR-21-5p and a 23 nucleotide long 5'-phosphorylated miRNA with 3'-uridylation (iso-miR-16-5p), were only detected in the CLL patients.

A chemometric approach for characterization of serum transthyretin in familial amyloidotic polyneuropathy type I (FAP-I) by electrospray ionization-ion mobility mass spectrometry.

In this study, we describe a chemometric data analysis approach to assist in the interpretation of the complex datasets from the analysis of high-molecular mass oligomeric proteins by ion mobility mass spectrometry (IM-MS). The homotetrameric protein transthyretin (TTR) is involved in familial amyloidotic polyneuropathy type I (FAP-I). FAP-I is associated with a specific TTR mutant variant (TTR(Met30)) that can be easily detected analyzing the monomeric forms of the mutant protein. However, the mechanism of protein misfolding and aggregation onset, which could be triggered by structural changes in the native tetrameric protein, remains under investigation. Serum TTR from healthy controls and FAP-I patients was purified under non-denaturing conditions by conventional immunoprecipitation in solution and analyzed by IM-MS. IM-MS allowed separation and characterization of several tetrameric, trimeric and dimeric TTR gas ions due to their differential drift time. After an appropriate data pre-processing, multivariate curve resolution alternating least squares (MCR-ALS) was applied to the complex datasets. A group of seven independent components being characterized by their ion mobility profiles and mass spectra were resolved to explain the observed data variance in control and patient samples. Then, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were considered for exploration and classification. Only four out of the seven resolved components were enough for an accurate differentiation. Furthermore, the specific TTR ions identified in the mass spectra of these components and the resolved ion mobility profiles provided a straightforward insight into the most relevant oligomeric TTR proteoforms for the disease.

Evaluation of ion mobility for the separation of glycoconjugate isomers due to different types of sialic acid linkage, at the intact glycoprotein, glycopeptide and glycan level.

The study of protein glycosylation can be regarded as an intricate but very important task, making glycomics one of the most challenging and interesting, albeit under-researched, type of "omics" science. Complexity escalates remarkably when considering that carbohydrates can form severely branched structures with many different constituents, which often leads to the formation of multiple isomers. In this regard, ion mobility (IM) spectrometry has recently demonstrated its power for the separation of isomeric compounds. In the present work, the potential of traveling wave IM (TWIMS) for the separation of isomeric glycoconjugates was evaluated, using mouse transferrin (mTf) as model glycoprotein. Particularly, we aim to assess the performance of this platform for the separation of isomeric glycoconjugates due to the type of sialic acid linkage, at the intact glycoprotein, glycopeptide and glycan level. Straightforward separation of isomers was achieved with the analysis of released glycans, as opposed to the glycopeptides which showed a more complex pattern. Finally, the developed methodology was applied to serum samples of mice, to investigate its robustness when analyzing real complex samples. BIOLOGICAL SIGNIFICANCE: Ion mobility mass spectrometry is a promising analytical technique for the separation of glycoconjugate isomers due to type of sialic acid linkage. The impact of such a small modification in the glycan structure is more evident in smaller analytes, reason why the analysis of free glycans was easier compared to the intact protein or the glycopeptides. The established methodology could be regarded as starting point in the separation of highly decorated glycoconjugates. This is an important topic nowadays, as differences in the abundance of some glycan isomers could be the key for the early diagnosis, control or differentiation of certain diseases, such as inflammation or cancer.

Determination of acidity constants and prediction of electrophoretic separation of amyloid beta peptides.

In this paper we describe a strategy to estimate by CE the acidity constants (pKa) of complex polyprotic peptides from their building peptide fragments. CE has been used for the determination of the pKas of five short polyprotic peptides that cover all the sequence of amyloid beta (Aß) peptides 1-40 and 1-42 (Aß fragments 1-15, 10-20, 20-29, 25-35 and 33-42). First, the electrophoretic mobility (me) was measured as a function of pH of the background electrolyte (BGE) in the pH range 2-12 (bare fused silica capillary, I=25mM and T=25°C). Second, the mes were fitted to equations modelling the ionisable behaviour of the different fragments as a function of pH to determine their pKas. The accuracy of the pKas was demonstrated predicting the electrophoretic behaviour of the studied fragments using the classical semiempirical relationships between me and peptide charge-to-mass ratio (me vs. q/Mr1/2, classical polymer model, q=charge and Mr=relative molecular mass). Separation selectivity in a mixture of the fragments as a function of pH was evaluated, taking into account the influence of the electroosmotic flow (EOF) at each pH value, and a method for the simple and rapid simulation of the electropherograms at the optimum separation pH was described. Finally, the pKas of the fragments were used to estimate the pKas of the Aß peptides 1-40 and 1-42 (tC and D 3.1, E 4.6 and Y 10.8 for acidic amino acids and tN-D 8.6, H 6.0, K 10.6 and R 12.5 for basic amino acids), which were used to predict their behaviour and simulate their electropherograms with excellent results. However, as expected due to the very small differences on q/Mr1/2 values, separation resolution of their mixtures was poor over the whole pH range. The use of poly(vinyl alcohol) (PVA) coated capillaries allowed reducing the EOF and a slight improvement of resolution.

On-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry using Fab´antibody fragments for the analysis of serum transthyretin.

This paper describes an on-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry (IA-SPE-CE-MS) method using an immunoaffinity sorbent with Fab' antibody fragments (Fab'-IA) for the analysis of serum transthyretin (TTR), a homotetrameric protein (Mr~56,000) involved in different types of amyloidosis. The IA sorbent was prepared by covalent attachment of Fab' fragments obtained from a polyclonal IgG antibody against TTR to succinimidyl silica particles. The Fab'-IA-SPE-CE-MS methodology was first established analyzing TTR standard solutions. Under optimized conditions, repeatability and reproducibility were acceptable, the method was linear between 1 and 25µgmL-1, limits of detection (LODs) were around 0.5µgmL-1 (50-fold lower than by CE-MS, ~25µgmL-1) and different TTR conformations were observed (folded and unfolded). The applicability of the developed method to screen for familial amyloidotic polyneuropathy type I (FAP-I), which is the most common hereditary systemic amyloidosis, was evaluated analyzing serum samples from healthy controls and FAP-I patients. For the analysis of sera, the most abundant proteins were precipitated with 5% (v/v) of phenol before Fab'-IA-SPE-CE-MS. The current method enhanced our previous results for the analysis of TTR using intact antibodies immobilized on magnetic beads. It allowed a slight improvement on LODs (2-fold), the detection of proteoforms found at lower concentrations and the preparation of microcartridges with extended durability.

Identification and characterization of isomeric N-glycans of human alfa-acid-glycoprotein by stable isotope labelling and ZIC-HILIC-MS in combination with exoglycosidase digestion.

In this study, a ZIC-HILIC-MS methodology for the analysis of N-glycan isomers was optimized to obtain greater detection sensitivity and thus identify more glycan structures in hAGP. In a second step, this method was combined with glycan reductive isotope labelling (GRIL) through [(12)C6]/[(13)C6]-aniline and exoglycosidase digestion to characterize the different glycan isomers. The GRIL method allows the peak areas resulting from two different labelled samples to be compared, since neither retention time shifts nor variations in the ionization of glycans between these samples are obtained. First, sialic acid linkage assignations were performed for most hAGP glycan isomers with α2-3 sialidase digestion. Bi-, tri- and tetraantennary glycan isomers with different terminal sialic acid linkages to galactose (α2-3 or α2-6) were assigned, and the potential of this technique for the structural characterization of isobaric isomers was therefore demonstrated. Furthermore, fucose linkage isomers of hAGP glycans were also characterized using this isotope-labelling approach in combination with α1-3,4 fucosidase and ß1-4 galactosidase digestion. α1-3 antennary fucoses and α1-6 core fucosylation were detected in hAGP fucosylated glycans. These established methodologies can be extremely useful for patho-glycomic studies to characterize glycoproteins of biomedical interest and find novel glycan isomers that could be used as biomarkers in cancer research.

On-Line Solid-Phase Extraction Capillary Electrophoresis Mass Spectrometry for Preconcentration and Clean-Up of Peptides and Proteins.

One of the major drawbacks of capillary electrophoresis (CE) and other microscale separation techniques, for the analysis of low abundant peptides and proteins in complex samples, are the poor concentration limits of detection. Several strategies have been developed to improve CE sensitivity. Here, we describe an on-line solid-phase extraction capillary electrophoresis mass spectrometry method with a commercial C18 sorbent for clean-up and preconcentration of neuropeptides from highly diluted biological samples.

Comparison of capillary electrophoresis and capillary liquid chromatography coupled to mass spectrometry for the analysis of transthyretin in human serum.

Capillary electrophoresis and capillary liquid chromatography coupled to mass spectrometry (CE-MS and CapLC-MS, respectively) are nowadays very suitable techniques for the separation and characterization of intact proteins in biological fluids. In this paper, we compare the performance of both techniques for the analysis of transthyretin (TTR), which is a homotetrameric protein (relative molecular mass (Mr) ∼56,000) involved in different types of amyloidosis. Furthermore, it is also presented a novel sample pretreatment based on immunoprecipitation (IP) using Protein A Ultrarapid Agarose™ (UAPA) magnetic beads (MBs) to purify TTR from serum samples. This novel IP based on MBs allowed the detection of TTR monomeric proteoforms that were not possible to analyze by conventional IP in solution. In addition, UAPA MBs provided many other desirable advantages including higher selectivity and minimal unspecific binding of other proteins. CE-MS and CapLC-MS were applied to analyze serum samples from healthy controls and familial amyloidotic polyneuropathy type I (FAP-I) patients, who suffered from the most common hereditary systemic amyloidosis. Both techniques allowed detecting the same TTR proteoforms, including the mutant TTR (Met 30) variant (variation in relative molecular mass (ΔMr) was +32.07, from wild-type TTR). Migration/retention times and relative quantitation of the different proteoforms were similar and reproducible in both cases, but the limits of detection (LODs) achieved by CE-MS were slightly lower (2-2.5-fold). Some other differences were also found on separation selectivity (migration orders and separation of antibody), peak efficiency, total analysis time, calibration ranges and experimental Mr accuracy.

Analysis of serum transthyretin by on-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry using magnetic beads.

In this paper, an on-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry (IA-SPE-CE-MS) method using magnetic beads (MBs) is described for the analysis of serum transthyretin (TTR), which is a protein related to different types of amyloidosis. First, purification of TTR from serum was investigated by off-line immunoprecipitation and CE-MS. The suitability of three Protein A (ProA) MBs (Protein A Ultrarapid Agarose(TM) (UAPA), Dynabeads(®) Protein A (DyPA) and SiMAG-Protein A (SiPA) and AffiAmino Ultrarapid Agarose(TM) (UAAF) MBs to prepare an IA sorbent with a polyclonal antibody (Ab) against TTR, was studied. In all cases, results were repeatable and it was possible the identification and the quantitation of the relative abundance of the six most abundant TTR proteoforms. Although recoveries were the best with UAPA MBs, UAAF MBs were preferred for on-line immunopurification because Ab was not eluted from the MBs. Under the optimized conditions with standards in IA-SPE-CE-MS, microcartridge lifetime (>20 analyses/day) and repeatability (2.9 and 4.3% RSD for migration times and peak areas) were good, the method was linear between 5 and 25 µg/mL and LOD was around 1 µg/mL (25 times lower than by CE-MS, ≈25 µg/mL). A simple off-line sample pretreatment based on precipitation of the most abundant proteins with 5% (v/v) of phenol was necessary to clean-up serum samples. The potential of the on-line method to screen for familial amyloidotic polyneuropathy type I (FAP-I), which is the most common hereditary systemic amyloidosis, was demonstrated analysing serum samples from healthy controls and FAP-I patients.

Increased α1-3 fucosylation of α-1-acid glycoprotein (AGP) in pancreatic cancer.

Pancreatic cancer (PDAC) lacks reliable diagnostic biomarkers and the search for new biomarkers represents an important challenge. Previous results looking at a small cohort of patients showed an increase in α-1-acid glycoprotein (AGP) fucosylation in advanced PDAC using N-glycan sequencing. Here, we have analysed AGP glycoforms in a larger cohort using several analytical techniques including mass spectrometry (MS), capillary zone electrophoresis (CZE) and enzyme-linked lectin assays (ELLAs) for determining AGP glycoforms which could be PDAC associated. AGP from 31 serum samples, including healthy controls (HC), chronic pancreatitis (ChrP) and PDAC patients, was purified by immunoaffinity chromatography. Stable isotope labelling of AGP released N-glycans and their analysis by zwitterionic hydrophilic interaction capillary liquid chromatography electrospray MS (µZIC-HILIC-ESI-MS) showed an increase in AGP fucosylated glycoforms in PDAC compared to ChrP and HC. By CZE-UV analysis, relative concentrations of some of the AGP isoforms were found significantly different compared to those in PDAC and HC. Finally, ELLAs using Aleuria aurantia lectin displayed a significant increase in AGP fucosylation, before and after AGP neuraminidase treatment, in advanced PDAC compared to ChrP and HC, respectively. Altogether, these results indicate that α1-3 fucosylated glycoforms of AGP are increased in PDAC and could be potentially regarded as a PDAC biomarker.

Characterisation of serum transthyretin by electrospray ionisation-ion mobility mass spectrometry: Application to familial amyloidotic polyneuropathy type I (FAP-I).

Transthyretin (TTR) is a homotetrameric protein which is known to misfold and aggregate causing different types of amyloidosis, such as familial amyloidotic polyneuropathy type I (FAP-I). FAP-I is associated with a specific TTR mutant variant (TTR (Met30)) that can be easily detected analysing the monomeric forms of the mutant protein. Meanwhile, the mechanism of protein aggregation onset, which could be triggered by structural changes on the native tetrameric protein complex, remains uncertain. We developed and described herein a new sample pretreatment based on immunoprecipitation (IP) to purify TTR from serum under non-denaturing conditions. Later, a nano-electrospray ionization-ion mobility mass spectrometry (nano-ESI-IM-MS or IM-MS) method was optimised to analyse the protein complexes in serum samples from healthy controls and FAP-I patients. IM-MS allowed separation and characterisation of tetrameric, trimeric and dimeric TTR gas ions due to their differential drift time, which is related to ion size and charge. The tetramer-to-dimer abundance ratio was differential between healthy controls and FAP-I patients (asymptomatic, symptomatic and an iatrogenic patient originally without the mutation who received a liver transplant from an FAP-I patient), and was also indicative of the effectiveness of liver transplantation as a treatment for FAP-I.

Pentacyclic triterpene in Olea europaea L: A simultaneous determination by high-performance liquid chromatography coupled to mass spectrometry.

Pentacyclic triterpenes are gaining interest due to their beneficial health effects, as anti-inflammatory, anti-diabetic and anti-tumoral, among others. In this study, an analytical LC-MS method was developed for the simultaneous determination of maslinic, oleanolic and ursolic acids along with erythrodiol and uvaol, which are the main triterpenic compounds present in the fruits and leaves of Olea europaea L. A Zorbax Eclipse PAH column at 30°C with mobile phase of water (17%) and methanol (83%) at 0.8mL/min conformed the optimal chromatographic conditions that allowed the separation of the compounds of interest, two pairs of which are isomers differing only in the position of one methyl group (oleanolic-ursolic and erythrodiol-uvaol). The ionization was performed in an APCI source at 450°C programmed in negative mode for the triterpenic acids, and in positive for the alcohols. An ion trap (LC-IT-MS) and a triple quadrupole (LC-QqQ-MS) were assessed for maximal sensitivity that was achieved with LC-QqQ-MS. The LODs of triterpenic acids were lower than 1nM, whereas for erythrodiol and uvaol were 4.5 and 7.5nM, respectively. The method was linear for the five analytes in the range of concentrations from 0.005 to 15µM with correlation coefficients exceeding 0.99. The precision and accuracy were ≤9.90% and ≤9.57%, respectively. The applicability of the validated method was assessed in the analysis of the pentacyclic triterpenes in Marfil table olives, after the optimization of the extraction procedure. The developed method constitutes the first step for future studies of triterpenic compounds present in foods that would allow establishing their effects on human health.

Analysis of transthyretin in human serum by capillary zone electrophoresis electrospray ionization time-of-flight mass spectrometry. Application to familial amyloidotic polyneuropathy type I.

Transthyretin (TTR) is known to misfold and aggregate, causing different types of amyloidosis. Familial amyloidotic polyneuropathy type I (FAP-I), which is the most common hereditary systemic amyloidosis, is associated with a TTR variant that presents a single amino acid substitution of valine for methionine at position 30 (Met 30). To screen for TTR-related amyloidosis rapidly and reliably, we have developed a novel procedure based on the analysis of monomers from the homotetrameric protein (∼56 kDa). First, we established a CZE-ESI-TOF-MS method to detect wild-type (normal) TTR with or without several PTMs, as well as an extra minor isoform in TTR standard solutions. Later, a sample pretreatment based on immunoprecipitation (IP) and centrifugal filtration was optimized to analyze serum samples from healthy controls and FAP-I patients (including an asymptomatic patient, a symptomatic patient, a liver-transplanted patient with the specific mutation, and a patient originally without the mutation who received a liver transplant from an FAP-I patient (iatrogenic FAP-I)). The mutant TTR (Met 30) variant with a relative molecular mass 32.07 higher than the wild-type TTR was found in the asymptomatic, the symptomatic and the iatrogenic FAP-I patients, who interestingly also presented the same concentration ratio between both variants of TTR (abnormal and normal). In contrast, as in the healthy controls, the abnormal TTR variant was not detected in the liver-transplanted patient with the specific mutation, which confirms the effectiveness of the treatment. The proposed procedure could be regarded as a suitable screening system for individuals with suspected TTR amyloidosis, and to gain insight into TTR structure, to understand the mechanism underlying the disease.

Quantitative analysis of N-glycans from human alfa-acid-glycoprotein using stable isotope labeling and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry as tool for pancreatic disease diagnosis.

In this work we demonstrate the potential of glycan reductive isotope labeling (GRIL) using [(12)C]- and [(13)C]-coded aniline and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry (µZIC-HILIC-ESI-MS) for relative quantitation of glycosylation variants in selected glycoproteins present in samples from cancer patients. Human α1-acid-glycoprotein (hAGP) is an acute phase serum glycoprotein whose glycosylation has been described to be altered in cancer and chronic inflammation. However, it is not clear yet whether some particular glycans in hAGP can be used as biomarker for differentiating between these two pathologies. In this work, hAGP was isolated by immunoaffinity chromatography (IAC) from serum samples of healthy individuals and from those suffering chronic pancreatitis and different stages of pancreatic cancer, respectively. After de-N-glycosylation, relative quantitation of the hAGP glycans was carried out using stable isotope labeling and µZIC-HILIC-ESI-MS analysis. First, protein denaturing conditions prior to PNGase F digestion were optimized to achieve quantitative digestion yields, and the reproducibility of the established methodology was evaluated with standard hAGP. Then, the proposed method was applied to the analysis of the clinical samples (control vs. pathological). Pancreatic cancer samples clearly showed an increase in the abundance of fucosylated glycans as the stage of the disease increases and this was unlike to samples from chronic pancreatitis. The results gained here indicate the mentioned glycan in hAGP as a candidate structure worth to be corroborated by an extended study including more clinical cases; especially those with chronic pancreatitis and initial stages of pancreatic cancer. Importantly, the results demonstrate that the presented methodology combining an enrichment of a target protein by IAC with isotope coded relative quantitation of N-glycans can be successfully used for targeted glycomics studies. The methodology is assumed being suitable as well for other such studies aimed at finding novel cancer associated glycoprotein biomarkers.