50 research outputs found
FASIL-MS: An Integrated Proteomic and Bioinformatic Workflow To Universally Quantitate In Vivo-Acetylated Positional Isomers
Dynamic
changes in histone post-translational modifications (PTMs)
regulate gene transcription leading to fine-tuning of biological processes
such as DNA replication and cell cycle progression. Moreover, specific
histone modifications constitute docking sites for recruitment of
DNA damage repair proteins and mediation of subsequent cell survival.
Therefore, understanding and monitoring changes in histone PTMs that
can alter cell proliferation and thus lead to disease progression
are of considerable medical interest. In this study, stable isotope
labeling with <i>N</i>-acetoxy-D<sub>3</sub>-succinimide
(D<sub>3</sub>-NAS) was utilized to efficiently derivatize unmodified
lysine residues at the protein level. The sample preparation method
was streamlined to facilitate buffer exchange between the multiple
steps of the protocol by coupling chemical derivatization to filter-aided
sample preparation (FASP). Additionally, the mass spectrometry method
was adapted to simultaneously coisolate and subsequently cofragment
all differentially H<sub>3</sub>/D<sub>3</sub>-acetylated histone
peptide clusters. Combination of these multiplexed MS<sup>2</sup> spectra
with the implementation of a data analysis algorithm enabled the quantitation
of each and every in vivo-acetylated DMSO- and SAHA-treated H4(4ā17)
and H3(18ā26) peptide. We have termed our new approach FASIL-MS
for filter-aided stable isotopic labeling coupled to mass spectrometry.
FASIL-MS enables the universal and site-specific quantitation of peptides
with multiple in vivo-acetylated lysine residues. Data are available
via ProteomeXchange (PXD003611)
Multiple and Sequential Data Acquisition Method: An Improved Method for Fragmentation and Detection of Cross-Linked Peptides on a Hybrid Linear Trap Quadrupole Orbitrap Velos Mass Spectrometer
The identification and validation of cross-linked peptides
by mass
spectrometry remains a daunting challenge for proteināprotein
cross-linking approaches when investigating protein interactions.
This includes the fragmentation of cross-linked peptides in the mass
spectrometer per se and following database searching, the matching
of the molecular masses of the fragment ions to the correct cross-linked
peptides. The hybrid linear trap quadrupole (LTQ) Orbitrap Velos combines
the speed of the tandem mass spectrometry (MS/MS) duty circle with
high mass accuracy, and these features were utilized in the current
study to substantially improve the confidence in the identification
of cross-linked peptides. An MS/MS method termed multiple and sequential
data acquisition method (MSDAM) was developed. Preliminary optimization
of the MS/MS settings was performed with a synthetic peptide (TP1)
cross-linked with bisĀ[sulfosuccinimidyl] suberate (BS<sup>3</sup>).
On the basis of these results, MSDAM was created and assessed on the
BS<sup>3</sup>-cross-linked bovine serum albumin (BSA) homodimer.
MSDAM applies a series of multiple sequential fragmentation events
with a range of different normalized collision energies (NCE) to the
same precursor ion. The combination of a series of NCE enabled a considerable
improvement in the quality of the fragmentation spectra for cross-linked
peptides, and ultimately aided in the identification of the sequences
of the cross-linked peptides. Concurrently, MSDAM provides confirmatory
evidence from the formation of reporter ions fragments, which reduces
the false positive rate of incorrectly assigned cross-linked peptides
Combining Filter-Aided Sample Preparation and Pseudoshotgun Technology To Profile the Proteome of a Low Number of Early Passage Human Melanoma Cells
The performance of two proteomic sample preparation methods,
āpseudoshotgunā
(PSG) and filter-aided sample preparation (FASP) were compared in
terms of the number of identified proteins, representation of cellular
component GO (gene ontology) categories in the obtained list of proteins,
and the efficiency of both methods in the proteomic analysis of a
very low number of cells. Both methods were combined to obtain a proteomic
profile of a short-term culture (passage 3) of melanoma cells, established
in our laboratory from a human metastatic melanoma lesion. The data
revealed that with FASP, usually more proteins are identified than
with PSG when analyzing a higher number of cells (ā„5000/injection),
whereas PSG is favorable when analyzing only a very small amount of
cells (250ā500/injection). PSG and FASP, however, are complementary
techniques, as combining both methods further increases the number
of identified proteins. Moreover, we show that it is feasible to identify
a substantial number of proteins from only 250 cells/injection that
is equivalent to 60 ng of protein
abFASP-MS: Affinity-Based Filter-Aided Sample Preparation Mass Spectrometry for Quantitative Analysis of Chemically Labeled Protein Complexes
Affinity
purification coupled to 1-D gel-free liquid chromatography
mass spectrometry (LCāMS) is a well-established and widespread
approach for the analyses of noncovalently interacting protein complexes.
In this study, two proteins conjugated to a streptavidin-binding peptide
and hemagglutinin double tag were expressed in the respective Flp-In
HEK293 cell lines: green fluorescent protein (SH-GFP) and TANK binding
kinase 1 (SH-TBK1_MOUSE). Fluorescent anti-HA immunoblots revealed
that the expression level of SH-GFP was ā¼50% lower than that
of SH-TBK1_MOUSE. Subsequently, the input material was normalized
to obtain a similar quantity of purified SH-tagged proteins. Optimization
of the release of protein complexes from the <i>anti</i>-HA-agarose with different eluting agents was then assessed. With
respect to the total number of protein groups identified in the purified
complexes, elution with 2% SDS surpassed both 100 mM glycine and 100
mM formic acid. Relative quantitation of the purified protein complexes
using TMT 6-plex reagents confirmed the higher efficiency of the 2%
SDS elution followed by filter-aided sample preparation (FASP). The
data presented in this study provide a new application of FASP to
quantitative MS analysis of affinity-purified protein complexes. We
have termed the approach abFASP-MS, or affinity-based filter-aided
sample preparation mass spectrometry
Combining Filter-Aided Sample Preparation and Pseudoshotgun Technology To Profile the Proteome of a Low Number of Early Passage Human Melanoma Cells
The performance of two proteomic sample preparation methods,
āpseudoshotgunā
(PSG) and filter-aided sample preparation (FASP) were compared in
terms of the number of identified proteins, representation of cellular
component GO (gene ontology) categories in the obtained list of proteins,
and the efficiency of both methods in the proteomic analysis of a
very low number of cells. Both methods were combined to obtain a proteomic
profile of a short-term culture (passage 3) of melanoma cells, established
in our laboratory from a human metastatic melanoma lesion. The data
revealed that with FASP, usually more proteins are identified than
with PSG when analyzing a higher number of cells (ā„5000/injection),
whereas PSG is favorable when analyzing only a very small amount of
cells (250ā500/injection). PSG and FASP, however, are complementary
techniques, as combining both methods further increases the number
of identified proteins. Moreover, we show that it is feasible to identify
a substantial number of proteins from only 250 cells/injection that
is equivalent to 60 ng of protein
abFASP-MS: Affinity-Based Filter-Aided Sample Preparation Mass Spectrometry for Quantitative Analysis of Chemically Labeled Protein Complexes
Affinity
purification coupled to 1-D gel-free liquid chromatography
mass spectrometry (LCāMS) is a well-established and widespread
approach for the analyses of noncovalently interacting protein complexes.
In this study, two proteins conjugated to a streptavidin-binding peptide
and hemagglutinin double tag were expressed in the respective Flp-In
HEK293 cell lines: green fluorescent protein (SH-GFP) and TANK binding
kinase 1 (SH-TBK1_MOUSE). Fluorescent anti-HA immunoblots revealed
that the expression level of SH-GFP was ā¼50% lower than that
of SH-TBK1_MOUSE. Subsequently, the input material was normalized
to obtain a similar quantity of purified SH-tagged proteins. Optimization
of the release of protein complexes from the <i>anti</i>-HA-agarose with different eluting agents was then assessed. With
respect to the total number of protein groups identified in the purified
complexes, elution with 2% SDS surpassed both 100 mM glycine and 100
mM formic acid. Relative quantitation of the purified protein complexes
using TMT 6-plex reagents confirmed the higher efficiency of the 2%
SDS elution followed by filter-aided sample preparation (FASP). The
data presented in this study provide a new application of FASP to
quantitative MS analysis of affinity-purified protein complexes. We
have termed the approach abFASP-MS, or affinity-based filter-aided
sample preparation mass spectrometry
Portfolio management of mixed-species forests
We propose to test the portfolio selection theory on the historical data of tree speciesā productivities obtained from the French National Forest Inventory (IFN). We determine the optimal timber productivity-vulnerability arrangements out of the combinations of tree species and map the optimal compositions per administrative department in France. We also estimate the survivals of optimal portfolios using the speciesā probabilities of presence. Our results show that greater weights in the optimal portfolios correspond to higher probabilities of presence
abFASP-MS: Affinity-Based Filter-Aided Sample Preparation Mass Spectrometry for Quantitative Analysis of Chemically Labeled Protein Complexes
Affinity
purification coupled to 1-D gel-free liquid chromatography
mass spectrometry (LCāMS) is a well-established and widespread
approach for the analyses of noncovalently interacting protein complexes.
In this study, two proteins conjugated to a streptavidin-binding peptide
and hemagglutinin double tag were expressed in the respective Flp-In
HEK293 cell lines: green fluorescent protein (SH-GFP) and TANK binding
kinase 1 (SH-TBK1_MOUSE). Fluorescent anti-HA immunoblots revealed
that the expression level of SH-GFP was ā¼50% lower than that
of SH-TBK1_MOUSE. Subsequently, the input material was normalized
to obtain a similar quantity of purified SH-tagged proteins. Optimization
of the release of protein complexes from the <i>anti</i>-HA-agarose with different eluting agents was then assessed. With
respect to the total number of protein groups identified in the purified
complexes, elution with 2% SDS surpassed both 100 mM glycine and 100
mM formic acid. Relative quantitation of the purified protein complexes
using TMT 6-plex reagents confirmed the higher efficiency of the 2%
SDS elution followed by filter-aided sample preparation (FASP). The
data presented in this study provide a new application of FASP to
quantitative MS analysis of affinity-purified protein complexes. We
have termed the approach abFASP-MS, or affinity-based filter-aided
sample preparation mass spectrometry
Evaluating the Promiscuous Nature of Tyrosine Kinase Inhibitors Assessed in A431 Epidermoid Carcinoma Cells by Both Chemical- and Phosphoproteomics
Deregulation
of protein tyrosine kinase signaling has been linked
to many diseases, most notably cancer. As a consequence, small molecule
inhibitors of protein tyrosine kinases may provide powerful strategies
for treatment. Following the successful introduction of imatinib in
the treatment of chronic myelogenous leukemia, such drugs are also
now evaluated for other types of cancer. However, many developed kinase
inhibitors are not very target-specific and therefore may induce side
effects. The importance of such side effects is certainly cell-proteome
dependent. Understanding the all-inclusive action of a tyrosine kinase
inhibitor on each individual cell-type entails the identification
of potential targets, combined with monitoring the downstream effects
revealing the signaling networks involved. Here, we explored a multilevel
quantitative mass spectrometryābased proteomic strategy to
identify the direct targets and downstream signaling effect of four
tyrosine kinase inhibitors (imatinib, dasatinib, bosutinib, and nilotinib)
in epidermoid carcinoma cells, as a model system for skin-cancer.
More than 25 tyrosine kinases showed affinity to the drugs, with imatinib
and nilotinib displaying a high specificity, especially when compared
to dasatinib and bosutinib. Consequently, the latter two drugs showed
a larger effect on downstream phosphotyrosine signaling. Many of the
proteins affected are key regulators in cell adhesion and invasion.
Our data represents a multiplexed view on the promiscuous action of
certain tyrosine kinase inhibitors that needs to be taking into consideration
prior to the application of these drugs in the treatment of different
forms of cancer
Affinity Purification Strategies for Proteomic Analysis of Transcription Factor Complexes
Affinity purification (AP) coupled
to mass spectrometry (MS) has
been successful in elucidating protein molecular networks of mammalian
cells. These approaches have dramatically increased the knowledge
of the interconnectivity present among proteins and highlighted biological
functions within different protein complexes. Despite significant
technical improvements reached in the past years, it is still challenging
to identify the interaction networks and the subsequent associated
functions of nuclear proteins such as transcription factors (TFs).
A straightforward and robust methodology is therefore required to
obtain unbiased and reproducible interaction data. Here we present
a new approach for TF AP-MS, exemplified with the CCAAT/enhancer binding
protein alpha (C/EBPalpha). Utilizing the advantages of a double tag
and three different MS strategies, we conducted a total of six independent
AP-MS strategies to analyze the proteināprotein interactions
of C/EBPalpha. The resultant data were combined to produce a cohesive
C/EBPalpha interactome. Our study describes a new methodology that
robustly identifies specific molecular complexes associated with transcription
factors. Moreover, it emphasizes the existence of TFs as protein complexes
essential for cellular biological functions and not as single, static
entities