On-line coupling of aptamer affinity solid-phase extraction and immobilized enzyme microreactor capillary electrophoresis-mass spectrometry for the sensitive targeted bottom-up analysis of protein biomarkers

In this paper, we present a fully integrated valve-free method for the sensitive targeted bottom-up analysis of proteins through on-line aptamer affinity solid-phase extraction and immobilized enzyme microreactor capillary electrophoresis-mass spectrometry (AA-SPE-IMER-CE-MS). The method was developed analyzing α-synuclein (α-syn), which is a protein biomarker related to different neurodegenerative disorders, including Parkinson's disease. Under optimized conditions, on-line purification and preconcentration of α-syn, enzymatic digestion, electrophoretic separation, and identification of the tryptic peptides by mass spectrometry was achieved in less than 35 min. The limit of detection was 0.02 μg mL-1 of digested protein (66.7% of coverage, i.e., 8 out of 12 expected tryptic peptides were detected). This value was 125 and 10 times lower than for independent on-line digestion by IMER-CE-MS (2.5 μg mL-1) and on-line preconcentration by AA-SPE-CE-MS (0.2 μg mL-1). The repeatability of AA-SPE-IMER-CE-MS was adequate (at 0.5 μg mL-1,% RSD ranged from 3.7 to 16.9% for peak areas and 3.5 to 7.7% for migration times of the tryptic peptides), and the modified capillary could be reused up to 10 analyses with optimum performance, similarly to IMER-CE-MS. The method was subsequently applied to the analysis of endogenous α-syn from red blood cell lysates. Ten α-syn tryptic peptides were detected (83.3% of coverage), enabling the characterization and localization of post-translational modifications of blood α-syn (i.e., N-terminal acetylation)

Evaluation of on-line solid-phase extraction capillary electrophoresis-mass spectrometry with a nanoliter valve for the analysis of peptide biomarkers

On-line solid-phase extraction capillary electrophoresis-mass spectrometry (SPE-CE-MS) is a powerful technique for high throughput sample clean-up and analyte preconcentration, separation, detection, and characterization. The most typical design due to its simplicity and low cost is unidirectional SPE-CE-MS. However, in this configuration, the sample volumes introduced by pressure depend on the dimensions of the separation capillary and some matrix components could be irreversibly adsorbed in its inner walls. Furthermore, in many cases, the requirements of on-line preconcentration are incompatible with the background electrolyte necessary for an efficient separation and sensitive MS detection. Here, we present SPE-CE-MS with a nanoliter valve (nvSPE-CE-MS) to overcome these drawbacks while keeping the design simple. The nvSPE-CE-MS system is operated with a single CE instrument and two capillaries for independent and orthogonal SPE preconcentration and CE separation, which are interfaced through an external and electrically isolated valve with a 20 nL sample loop. The instrumental setup is proved for the analysis of opioid and amyloid beta peptide biomarkers in standards and plasma samples. NvSPE-CE-MS allowed decreasing the limits of detection (LODs) 200 times with regard to CE-MS. Compared to unidirectional SPE-CE-MS, peak efficiencies were better and repeatabilities similar, but total analysis times longer and LODs for standards slightly higher due to the heart-cut operation and the limited volume of the valve loop. This small difference on the LODs for standards was compensated for plasma samples by the improved tolerance of nvSPE-CE-MS to complex sample matrices. In view of these results, the presented setup can be regarded as a promising versatile alternative to avoid complicated matrix samples entering the separation capillary in SPE-CE-MS

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 (Aβ) peptides 1-40 and 1-42 (Aβ 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=25ºC). 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 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 Aβ 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 electroosmotic flow (EOF) and a slight improvement of resolution

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 review of sample preparation for purification of microRNAs and analysis by mass spectrometry methods

MicroRNAs (miRNAs) play an important role in regulation of different bioprocesses, including multiple diseases, such as cancer, neurodegenerative and immune-related disorders. Analysis of miRNA biomarkers in biological fluids requires accurate, sensitive, reproducible, and multiplexed methods. This review covers miRNA purification and measurement, which are the core of these analytical methods, and critically affect the output of biomarker research studies. With regard to miRNA measurement, the typical bioanalytical methods (e.g. reverse transcription polymerase chain reaction, RT-PCR), which have been extensively reviewed elsewhere, have been excluded to focus on less conventional methods based on mass spectrometry (MS). This review provides a broad overview of liquid-phase and solid-phase extraction purification methods for miRNA clean-up and enrichment and a critical insight into direct and indirect MS-based methods to disclose the true potential of MS in the field

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 AgaroseTM (UAPA), Dynabeads® Protein A (DyPA) and SiMAG-Protein A (SiPA)) and AffiAmino Ultrarapid AgaroseTM (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 6 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 optimised 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- 25 µg·mL-1 and limit of detection (LOD) was around 1 µg·mL-1 (25 times lower than by CE-MS, 25 µg·mL-1). 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

Risk factors associated with the antimicrobial resistance of staphylococci in canine pyoderma

This study reports the susceptibility to antimicrobial agents of staphylococci 4 (n=105) isolated from dogs, and the factors associated with this resistance. The study 5 animals were 23 healthy dogs (group A), 24 with first-time pyoderma (group B), and 27 6 with recurrent pyoderma that had undergone long-term antibiotic treatment (group C). 7 Staphylococci were more commonly isolated from the pyoderma-affected than the 8 healthy dogs (p<0.0001). 9 10 Some 78% of the isolates were resistant to at least one antimicrobial agent. 11 Resistance to amoxicillin-clavulanate,cephalosporins (OR 4.29, 95% CI [1.15, 16.3] 12 respectively), enrofloxacin (OR 9.47, 95% CI [1.53, 58.5]) and ciprofloxacin (OR 79.7 13 95% CI [3.26, 1947.4]) was more common among group C isolates. Some 32% of all 14 the isolates were multiresistant (MR) and 10.4% were methicillin-resistant (MRS). The 15 probability of isolating MRS staphylococci in group C increased by a factor of four 16 (95% CI [1.18, 17.9]) compared to A plus B. Multi-resistant (MR) isolates were 17 obtained more commonly from urban than rural dogs (OR 3.79, 95% CI [1.09, 13.17]). 18 All the MRS staphylococci encountered were obtained from urban dogs and more 19 commonly from male dogs (p= 0.07). 20 21 This study shows that dogs bred in urban habitat, with a history of antibiotic 22 therapy in the past year represents significant risk of being carriers of isolates resistant 23 to methicillin (MRS) and other antimicrobials. These factors should be considered 24 before applying an antimicrobial treatment in veterinary clinics

Identification of new immunoprotective surface protein vaccine candidates against streptococcus suis infection by proteomics

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