18 research outputs found
Integrated Quantification and Identification of Aldehydes and Ketones in Biological Samples
The
identification of unknown compounds remains to be a bottleneck of
mass spectrometry (MS)-based metabolomics screening experiments. Here,
we present a novel approach which facilitates the identification and
quantification of analytes containing aldehyde and ketone groups in
biological samples by adding chemical information to MS data. Our
strategy is based on rapid autosampler-in-needle-derivatization with <i>p</i>-toluenesulfonylhydrazine (TSH). The resulting TSH-hydrazones
are separated by ultrahigh-performance liquid chromatography (UHPLC)
and detected by electrospray ionization-quadrupole-time-of-flight
(ESI-QqTOF) mass spectrometry using a SWATH (Sequential Window Acquisition
of all Theoretical Fragment-Ion Spectra) data-independent high-resolution
mass spectrometry (HR-MS) approach. Derivatization makes small, poorly
ionizable or retained analytes amenable to reversed phase chromatography
and electrospray ionization in both polarities. Negatively charged
TSH-hydrazone ions furthermore show a simple and predictable fragmentation
pattern upon collision induced dissociation, which enables the chemo-selective
screening for unknown aldehydes and ketones via a signature fragment
ion (<i>m/z</i> 155.0172). By means of SWATH, targeted and
nontargeted application scenarios of the suggested derivatization
route are enabled in the frame of a single UHPLC-ESI-QqTOF-HR-MS workflow.
The methodâs ability to simultaneously quantify and identify
molecules containing aldehyde and ketone groups is demonstrated using
61 target analytes from various compound classes and a <sup>13</sup>C labeled yeast matrix. The identification of unknowns in biological
samples is detailed using the example of indole-3-acetaldehyde
Affimers as an Alternative to Antibodies in an Affinity LCâMS Assay for Quantification of the Soluble Receptor of Advanced Glycation End-Products (sRAGE) in Human Serum
Antibodies are indispensable tools
in biomedical research, but
their size, complexity, and sometimes lack of reproducibility created
a need for the development of alternative binders to overcome these
limitations. Affimers are a novel class of affinity binders based
on a structurally robust protease inhibitor scaffold (i.e., Cystatin
A), which are selected by phage display and produced in a rapid and
simple <i>E. coli</i> protein expression system. These binders
have a defined amino acid sequence with defined binding regions and
are versatile, thereby allowing for easy engineering. Here we present
an affimer-based liquid chromatographyâmass spectrometry (LCâMS)
method for quantification of the soluble Receptor of Advanced Glycation
End-products (sRAGE), a promising biomarker for chronic obstructive
pulmonary disease. The method was validated according to European
Medicines Agency and U.S. Food and Drug Administration guidelines
and enabled quantitation of serum sRAGE between 0.2 and 10 ng/mL.
Comparison between the affimer-based method and a previously developed,
validated antibody-based method showed good correlation (<i>R</i><sup>2</sup> = 0.88) and indicated that 25% lower sRAGE levels are
reported by the affimer-based assay. In conclusion, we show the first-time
application of affimers in a quantitative LCâMS method, which
supports the potential of affimers as robust alternatives to antibodies
Boron-Doped Diamond Electrodes for the Electrochemical Oxidation and Cleavage of Peptides
Electrochemical oxidation of peptides
and proteins is traditionally
performed on carbon-based electrodes. Adsorption caused by the affinity
of hydrophobic and aromatic amino acids toward these surfaces leads
to electrode fouling. We compared the performance of boron-doped diamond
(BDD) and glassy carbon (GC) electrodes for the electrochemical oxidation
and cleavage of peptides. An optimal working potential of 2000 mV
was chosen to ensure oxidation of peptides on BDD by electron transfer
processes only. Oxidation by electrogenerated OH radicals took place
above 2500 mV on BDD, which is undesirable if cleavage of a peptide
is to be achieved. BDD showed improved cleavage yield and reduced
adsorption for a set of small peptides, some of which had been previously
shown to undergo electrochemical cleavage C-terminal to tyrosine (Tyr)
and tryptophan (Trp) on porous carbon electrodes. Repeated oxidation
with BDD electrodes resulted in progressively lower conversion yields
due to a change in surface termination. Cathodic pretreatment of BDD
at a negative potential in an acidic environment successfully regenerated
the electrode surface and allowed for repeatable reactions over extended
periods of time. BDD electrodes are a promising alternative to GC
electrodes in terms of reduced adsorption and fouling and the possibility
to regenerate them for consistent high-yield electrochemical cleavage
of peptides. The fact that OH-radicals can be produced by anodic oxidation
of water at elevated positive potentials is an additional advantage
as they allow another set of oxidative reactions in analogy to the
Fenton reaction, thus widening the scope of electrochemistry in protein
and peptide chemistry and analytics
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
A Panel of Regulated Proteins in Serum from Patients with Cervical Intraepithelial Neoplasia and Cervical Cancer
We
developed a discoveryâvalidation mass-spectrometry-based
pipeline to identify a set of proteins that are regulated in serum
of patients with cervical intraepithelial neoplasia (CIN) and squamous
cell cervical cancer using iTRAQ, label-free shotgun, and targeted
mass-spectrometric quantification. In the discovery stage we used
a âpoolingâ strategy for the comparative analysis of
immunodepleted serum and revealed 15 up- and 26 down-regulated proteins
in patients with early- (CES) and late-stage (CLS) cervical cancer.
The analysis of nondepleted serum samples from patients with CIN,
CES, an CLS and healthy controls showed significant changes in abundance
of alpha-1-acid glycoprotein 1, alpha-1-antitrypsin, serotransferrin,
haptoglobin, alpha-2-HS-glycoprotein, and vitamin D-binding protein.
We validated our findings using a fast UHPLC/MRM method in an independent
set of serum samples from patients with cervical cancer or CIN and
healthy controls as well as serum samples from patients with ovarian
cancer (more than 400 samples in total). The panel of six proteins
showed 67% sensitivity and 88% specificity for discrimination of patients
with CIN from healthy controls, a stage of the disease where current
protein-based biomarkers, for example, squamous cell carcinoma antigen
(SCCA), fail to show any discrimination. Additionally, combining the
six-protein panel with SCCA improves the discrimination of patients
with CES and CLS from healthy controls
A Panel of Regulated Proteins in Serum from Patients with Cervical Intraepithelial Neoplasia and Cervical Cancer
We
developed a discoveryâvalidation mass-spectrometry-based
pipeline to identify a set of proteins that are regulated in serum
of patients with cervical intraepithelial neoplasia (CIN) and squamous
cell cervical cancer using iTRAQ, label-free shotgun, and targeted
mass-spectrometric quantification. In the discovery stage we used
a âpoolingâ strategy for the comparative analysis of
immunodepleted serum and revealed 15 up- and 26 down-regulated proteins
in patients with early- (CES) and late-stage (CLS) cervical cancer.
The analysis of nondepleted serum samples from patients with CIN,
CES, an CLS and healthy controls showed significant changes in abundance
of alpha-1-acid glycoprotein 1, alpha-1-antitrypsin, serotransferrin,
haptoglobin, alpha-2-HS-glycoprotein, and vitamin D-binding protein.
We validated our findings using a fast UHPLC/MRM method in an independent
set of serum samples from patients with cervical cancer or CIN and
healthy controls as well as serum samples from patients with ovarian
cancer (more than 400 samples in total). The panel of six proteins
showed 67% sensitivity and 88% specificity for discrimination of patients
with CIN from healthy controls, a stage of the disease where current
protein-based biomarkers, for example, squamous cell carcinoma antigen
(SCCA), fail to show any discrimination. Additionally, combining the
six-protein panel with SCCA improves the discrimination of patients
with CES and CLS from healthy controls
Antibody-Free LC-MS/MS Quantification of rhTRAIL in Human and Mouse Serum
The major challenge in targeted protein
quantification by LC-MS/MS
in serum lies in the complexity of the biological matrix with regard
to the wide diversity of proteins and their extremely large dynamic
concentration range. In this study, an LC-MS/MS method was developed
for the simultaneous quantification of the 60-kDa biopharmaceutical
proteins recombinant human tumor necrosis factor-related apoptosis-inducing
ligand wild type (rhTRAIL<sup>WT</sup>) and its death receptor 4 (DR4)-specific
variant rhTRAIL<sup>4C7</sup> in human and mouse serum. Selective
enrichment of TRAIL was accomplished by immobilized metal affinity
chromatography (IMAC), which was followed by tryptic digestion of
the enriched sample and quantification of a suitable signature peptide.
For absolute quantification, <sup>15</sup>N-metabolically labeled
internal standards of rhTRAIL<sup>WT</sup> and rhTRAIL<sup>4C7</sup> were used. Since the signature peptides that provided the highest
sensitivity and allowed discrimination between rhTRAIL<sup>WT</sup> and rhTRAIL<sup>4C7</sup> contained methionine residues, we oxidized
these quantitatively to their sulfoxides by the addition of 0.25%
(w/w) hydrogen peroxide. The final method has a lower limit of quantification
of 20 ng/mL (ca. 350 pM) and was fully validated according to current
international guidelines for bioanalysis. To show the applicability
of the LC-MS/MS method for pharmacokinetic studies, we quantified
rhTRAIL<sup>WT</sup> and rhTRAIL<sup>4C7</sup> simultaneously in serum
from mice injected intraperitoneally at a dose of 5 mg/kg for each
protein. This is the first time that two variants of rhTRAIL differing
by only a few amino acids have been analyzed simultaneously in serum,
an approach that is not possible by conventional enzyme-linked immuno-sorbent
assay (ELISA) analysis
Identification of Analytical Factors Affecting Complex Proteomics Profiles Acquired in a Factorial Design Study with Analysis of Variance: Simultaneous Component Analysis
Complex shotgun proteomics peptide
profiles obtained in quantitative
differential protein expression studies, such as in biomarker discovery,
may be affected by multiple experimental factors. These preanalytical
factors may affect the measured protein abundances which in turn influence
the outcome of the associated statistical analysis and validation.
It is therefore important to determine which factors influence the
abundance of peptides in a complex proteomics experiment and to identify
those peptides that are most influenced by these factors. In the current
study we analyzed depleted human serum samples to evaluate experimental
factors that may influence the resulting peptide profile such as the
residence time in the autosampler at 4 °C, stopping or not stopping
the trypsin digestion with acid, the type of blood collection tube,
different hemolysis levels, differences in clotting times, the number
of freezeâthaw cycles, and different trypsin/protein ratios.
To this end we used a two-level fractional factorial design of resolution
IV (2<sub>IV</sub><sup>7â3</sup>). The design required analysis of 16 samples in which the main effects
were not confounded by two-factor interactions. Data preprocessing
using the Threshold Avoiding Proteomics Pipeline (Suits, F.; Hoekman, B.; Rosenling, T.; Bischoff,
R.; Horvatovich, P. Anal. Chem. 2011, 83, 7786â7794, ref ) produced a data-matrix containing quantitative information on 2âŻ559
peaks. The intensity of the peaks was log-transformed, and peaks having
intensities of a low <i>t</i>-test significance (<i>p</i>-value > 0.05) and a low absolute fold ratio (<2)
between
the two levels of each factor were removed. The remaining peaks were
subjected to analysis of variance (ANOVA)-simultaneous component analysis
(ASCA). Permutation tests were used to
identify which of the preanalytical factors influenced the abundance
of the measured peptides most significantly. The most important preanalytical
factors affecting peptide intensity were (1) the hemolysis level,
(2) stopping trypsin digestion with acid, and (3) the trypsin/protein
ratio. This provides guidelines for the experimentalist to keep the
ratio of trypsin/protein constant and to control the trypsin reaction
by stopping it with acid at an accurately set pH. The hemolysis level
cannot be controlled tightly as it depends on the status of a patientâs
blood (e.g., red blood cells are more fragile in patients undergoing
chemotherapy) and the care with which blood was sampled (e.g., by
avoiding shear stress). However, its level can be determined with
a simple UV spectrophotometric measurement and samples with extreme
levels or the peaks affected by hemolysis can be discarded from further
analysis. The loadings of the ASCA model led to peptide peaks that
were most affected by a given factor, for example, to hemoglobin-derived
peptides in the case of the hemolysis level. Peak intensity differences
for these peptides were assessed by means of extracted ion chromatograms
confirming the results of the ASCA model
ELISA results of individual epithelial lining fluid (ELF) samples of young susceptible individuals, young non-susceptible individuals, and established COPD patients, before and after acute smoking.
<p>Results are given in box-plots with medians and interquartile ranges. *: p<0.05 before vs after smoking, â§: p<0.05 vs young susceptible individuals at baseline.</p
Summary of iTRAQ comparisons from pooled ELF samples.
<p>Summary of iTRAQ comparisons from pooled ELF samples.</p