15 research outputs found
Improvement of the Performance of Targeted LCâMS Assays through Enrichment of Histidine-Containing Peptides
Mass spectrometric-based quantification
using targeted methods
has matured during the past decade and is now commonly used in proteomics.
However, the reliability of protein quantification in complex matrixes
using selected reaction monitoring is often impaired by interfering
signals arising from coelution of nontargeted components. Sample preparation
methods resulting in the reduction of the number of peptides present
in the mixture minimizes this effect. One solution consists in the
selective capture of peptides containing infrequent amino acids. The
enrichment of histidine-containing peptides via immobilized metal-ion
affinity chromatography loaded with Cu<sup>2+</sup> ions (IMAC-Cu)
was applied in a quantitative workflow and found to be a simple and
cost effective method for the reduction of sample complexity with
high recovery and selectivity. When applied to a series of depleted
human plasma digests, the method decreased nonspecific signals, resulting
in a more precise and robust protein quantification. The method was
also shown to be an alternative to HSA/IgG depletion during plasma
protein analysis. This method, used in conjunction with recent improvements
in the instrumentâs peak capacity, addresses a bottleneck generally
encountered in quantitative proteomics studies by providing the robustness
and throughput required for the analysis of large sample series without
compromising the number of proteins monitored
Improvement of the Performance of Targeted LCâMS Assays through Enrichment of Histidine-Containing Peptides
Mass spectrometric-based quantification
using targeted methods
has matured during the past decade and is now commonly used in proteomics.
However, the reliability of protein quantification in complex matrixes
using selected reaction monitoring is often impaired by interfering
signals arising from coelution of nontargeted components. Sample preparation
methods resulting in the reduction of the number of peptides present
in the mixture minimizes this effect. One solution consists in the
selective capture of peptides containing infrequent amino acids. The
enrichment of histidine-containing peptides via immobilized metal-ion
affinity chromatography loaded with Cu<sup>2+</sup> ions (IMAC-Cu)
was applied in a quantitative workflow and found to be a simple and
cost effective method for the reduction of sample complexity with
high recovery and selectivity. When applied to a series of depleted
human plasma digests, the method decreased nonspecific signals, resulting
in a more precise and robust protein quantification. The method was
also shown to be an alternative to HSA/IgG depletion during plasma
protein analysis. This method, used in conjunction with recent improvements
in the instrumentâs peak capacity, addresses a bottleneck generally
encountered in quantitative proteomics studies by providing the robustness
and throughput required for the analysis of large sample series without
compromising the number of proteins monitored
Protein Quantification Using a Cleavable Reporter Peptide
Peptide
and protein quantification based on isotope dilution and
mass spectrometry analysis are widely employed for the measurement
of biomarkers and in system biology applications. The accuracy and
reliability of such quantitative assays depend on the quality of the
stable-isotope labeled standards. Although the quantification using
stable-isotope labeled peptides is precise, the accuracy of the results
can be severely biased by the purity of the internal standards, their
stability and formulation, and the determination of their concentration.
Here we describe a rapid and cost-efficient method to recalibrate
stable isotope labeled peptides in a single LCâMS analysis.
The method is based on the equimolar release of a protein reference
peptide (used as surrogate for the protein of interest) and a universal
reporter peptide during the trypsinization of a concatenated polypeptide
standard. The quality and accuracy of data generated with such concatenated
polypeptide standards are highlighted by the quantification of two
clinically important proteins in urine samples and compared with results
obtained with conventional stable isotope labeled reference peptides.
Furthermore, the application of the UCRP standards in complex samples
is described
A Simple Protocol To Routinely Assess the Uniformity of Proteomics Analyses
Mass-spectrometry-based
proteomic approaches are increasingly applied
to biological and clinical studies. Initially used by specialized
laboratories, the technology has matured and gained acceptance by
the community, using various analytical processes and platforms. To
facilitate data comparison and integration across laboratories, there
is a need to harmonize analytical processes to ensure the generation
of reliable proteomic data sets. This is especially critical in the
context of large initiatives, such as the <i>Human Proteome Project</i> promoted by the Human Proteome Organization (HUPO). Quality control
is a first step toward the harmonization of proteomics data sets.
We have developed a procedure to routinely assess the uniformity of
proteomics analyses. It relies on a simple protocol based on three
proteins and two sets of isotopically labeled peptides, one being
added prior to tryptic digestion and the second one prior to liquid
chromatographyâmass spectrometry (LCâMS) analysis. The
proposed method evaluates in a single step both the sample preparation,
by measuring the relative amounts of endogenous peptides and their
isotopically labeled counterparts, and the LCâMS platform performance,
by monitoring the main LCâMS attributes for reference peptides.
The procedure is simple and easy to implement into routine workflows
typically employed by the proteomics community
Longitudinal Urinary Protein Variability in Participants of the Space Flight Simulation Program
Urine
is a valuable material for the diagnosis of renal pathologies
and to investigate the effects of their treatment. However, the variability
in protein abundance in the context of normal homeostasis remains
a major challenge in urinary proteomics. In this study, the analysis
of urine samples collected from healthy individuals, rigorously selected
to take part in the <i>MARS-500</i> spaceflight simulation
program, provided a unique opportunity to estimate normal concentration
ranges for an extended set of urinary proteins. In order to systematically
identify and reliably quantify peptides/proteins across a large sample
cohort, a targeted mass spectrometry method was developed. The performance
of parallel reaction monitoring (PRM) analyses was improved by implementing
tight control of the monitoring windows during LCâMS/MS runs,
using an on-the-fly correction routine. Matching the experimentally
obtained MS/MS spectra with reference fragmentation patterns allowed
dependable peptide identifications to be made. Following optimization
and evaluation, the targeted method was applied to investigate protein
abundance variability in 56 urine samples, collected from six volunteers
participating in the <i>MARS-500</i> program. The intrapersonal
protein concentration ranges were determined for each individual and
showed unexpectedly high abundance variation, with an average difference
of 1 order of magnitude
Peptides Quantification by Liquid Chromatography with Matrix-Assisted Laser Desorption/Ionization and Selected Reaction Monitoring Detection
We present a novel analytical platform for peptides quantitative
assays in biological matrices based on microscale liquid chromatography
fractionation and matrix-assisted laser desorption/ionization mass
spectrometric detection using the selected reaction monitoring (SRM)
mode. The MALDI source was equipped with a high frequency Nd:YAG laser
(1000 Hz) and mounted on a triple quadrupole/linear ion trap mass
spectrometer (MALDI-QqQ<sub>LIT</sub>). Compared to conventional LCâESI-SRM/MS,
the separated analytes are âtime-frozenâ onto the MALDI
plate in fractions, and navigation through the LC chromatogram makes
it possible to perform SRM experiments as well as enhanced product
ion spectra acquisition for confirmatory analyses without time constraints.
The LC spots were analyzed using different rastering speeds ranging
from 0.25 to 4 mm/sec with the shortest analysis time of 425 ms/spot.
Since the LC runs can be multiplexed and do not need a comprehensive
investigation, the present platform offers a valuable alternative
to LCâESI-SRM/MS for high throughput proteomic analyses. In
addition, the derivatization of the N-terminal α-amino group
by sulfonation was found to be key for the fragmentation of singly
charged peptides under low collision energy regime. Under such conditions, <i>y</i>-ion series were observed in the MS/MS spectra, and thus
the design of SRM experiments was greatly simplified. The quantitative
performance of the platform was compared to that of LCâESI-SRM/MS
by spiking yeast tryptic peptides in human plasma digests. Both platforms
exhibited similar sensitivities, accuracy (within ±20%) and precision
(under 20%) in the relative quantification mode. As a proof of principle,
the relative and absolute quantification of proteins associated with
glycolysis, glyoxylate and tricarboxylic acid (TCA) cycles over a
growth time course of <i>Saccharomyces cerevisiae</i> on glucose media was successfully performed using isotopic
dilution
Biochemical and Physical Characterisation of Urinary Nanovesicles following CHAPS Treatment
<div><p>Urinary exosomes represent a precious source of potential biomarkers for disease biology. Currently, the methods for vesicle isolation are severely restricted by the tendency of vesicle entrapment, <em>e.g.</em> by the abundant Tamm-Horsfall protein (THP) polymers. Treatment by reducing agents such as dithiothreitol (DTT) releases entrapped vesicles, thus increasing the final yield. However, this harsh treatment can cause remodelling of all those proteins which feature extra-vesicular domains stabilized by internal disulfide bridges and have detrimental effects on their biological activity. In order to optimize exosomal yield, we explore two vesicle treatment protocols - dithiothreitol (DTT) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic (CHAPS) - applied to the differential centrifugation protocol for exosomal vesicle isolation. The results show that CHAPS treatment does not affect vesicle morphology or exosomal marker distribution, thus eliminating most of THP interference. Moreover, the recovery and preservation of catalytic activity of two trans-membrane proteases, dipeptidyl peptidase IV and nephrilysin, was examined and found to be clearly superior after CHAPS treatment compared to DTT. Finally, proteomic profiling by mass spectrometry (MS) revealed that 76.2% of proteins recovered by CHAPS are common to those seen for DTT treatment, which illustrates underlining similarities between the two approaches. In conclusion, we provide a major improvement to currently-utilized urinary vesicle isolation strategies to allow recovery of urinary vesicles without the deleterious interference of abundant urinary proteins, while preserving typical protein folding and, consequently, the precious biological activity of urinary proteins which serve as valuable biomarkers.</p> </div
TEM analysis.
<p>Transmission electron micrographs of <b>P18</b> (Panel A) and <b>P200</b> (Panel B) at 10,000Ă and 5,000Ă magnifications, respectively. High-magnification (50,000Ă) of CHAPS- (Panels C-F) and DTT-treated (Panels G-I) vesicle preparations are represented.</p
Protein identification in DTT and CHAPS supernatant.
<p>Partial list of proteins not previously reported in urinary exosomes and in 200,000 g supernatants.</p>a<p>Unique peptides on the total number of peptides.</p
Protein identification comparisons.
<p>Venn diagram showing the distribution of the number of identified proteins presents in SN 200,000 g after CHAPS and DTT treatments. Protein identifications from the current study were compared to two other studies which were carried out using high-resolution mass spectrometers in gels on 200,000 g pellets after DTT treatment (Gonzales et al. 2008) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037279#pone.0037279-Gonzales1" target="_blank">[5]</a> and 200,000 g supernatants (Kentsis et al. 2009) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037279#pone.0037279-Kentsis1" target="_blank">[23]</a>.</p