789 research outputs found
Fast Proteome Identification and Quantification from Data-Dependent Acquisition - Tandem Mass Spectrometry using Free Software Tools
Identification of nearly all proteins in a system using data-dependent
acquisition (DDA) mass spectrometry has become routine for simple organisms,
such as bacteria and yeast. Still, quantification of the identified proteins
may be a complex process and require multiple different software packages. This
protocol describes identification and label-free quantification of proteins
from bottom-up proteomics experiments. This method can be used to quantify all
the detectable proteins in any DDA dataset collected with high-resolution
precursor scans. This protocol may be used to quantify proteome remodeling in
response to a drug treatment or a gene knockout. Notably, the method uses the
latest and fastest freely-available software, and the entire protocol can be
completed in a few hours with data from organisms with relatively small
genomes, such as yeast or bacteria
Site-specific identification and quantitation of endogenous SUMO modifications under native conditions.
Small ubiquitin-like modifier (SUMO) modification regulates numerous cellular processes. Unlike ubiquitin, detection of endogenous SUMOylated proteins is limited by the lack of naturally occurring protease sites in the C-terminal tail of SUMO proteins. Proteome-wide detection of SUMOylation sites on target proteins typically requires ectopic expression of mutant SUMOs with introduced tryptic sites. Here, we report a method for proteome-wide, site-level detection of endogenous SUMOylation that uses α-lytic protease, WaLP. WaLP digestion of SUMOylated proteins generates peptides containing SUMO-remnant diglycyl-lysine (KGG) at the site of SUMO modification. Using previously developed immuno-affinity isolation of KGG-containing peptides followed by mass spectrometry, we identified 1209 unique endogenous SUMO modification sites. We also demonstrate the impact of proteasome inhibition on ubiquitin and SUMO-modified proteomes using parallel quantitation of ubiquitylated and SUMOylated peptides. This methodological advancement enables determination of endogenous SUMOylated proteins under completely native conditions
Comprehensive Overview of Bottom-up Proteomics using Mass Spectrometry
Proteomics is the large scale study of protein structure and function from
biological systems through protein identification and quantification. "Shotgun
proteomics" or "bottom-up proteomics" is the prevailing strategy, in which
proteins are hydrolyzed into peptides that are analyzed by mass spectrometry.
Proteomics studies can be applied to diverse studies ranging from simple
protein identification to studies of proteoforms, protein-protein interactions,
protein structural alterations, absolute and relative protein quantification,
post-translational modifications, and protein stability. To enable this range
of different experiments, there are diverse strategies for proteome analysis.
The nuances of how proteomic workflows differ may be challenging to understand
for new practitioners. Here, we provide a comprehensive overview of different
proteomics methods to aid the novice and experienced researcher. We cover from
biochemistry basics and protein extraction to biological interpretation and
orthogonal validation. We expect this work to serve as a basic resource for new
practitioners in the field of shotgun or bottom-up proteomics
SUCLA2 mutations cause global protein succinylation contributing to the pathomechanism of a hereditary mitochondrial disease
Mitochondrial acyl-coenzyme A species are emerging as important sources of protein modification and damage. Succinyl-CoA ligase (SCL) deficiency causes a mitochondrial encephalomyopathy of unknown pathomechanism. Here, we show that succinyl-CoA accumulates in cells derived from patients with recessive mutations in the tricarboxylic acid cycle (TCA) gene succinyl-CoA ligase subunit-beta (SUCLA2), causing global protein hyper-succinylation. Using mass spectrometry, we quantify nearly 1,000 protein succinylation sites on 366 proteins from patient-derived fibroblasts and myotubes. Interestingly, hyper-succinylated proteins are distributed across cellular compartments, and many are known targets of the (NAD(+))-dependent desuccinylase SIRT5. To test the contribution of hyper-succinylation to disease progression, we develop a zebrafish model of the SCL deficiency and find that SIRT5 gain-of-function reduces global protein succinylation and improves survival. Thus, increased succinyl-CoA levels contribute to the pathology of SCL deficiency through post-translational modifications. The pathomechanism of succinyl-CoA ligase (SCL) deficiency, a hereditary mitochondrial disease, is not fully understood. Here, the authors show that increased succinyl-CoA levels contribute to SCL pathology by causing global protein hyper-succinylation.Peer reviewe
Swift follow-up observations of candidate gravitational-wave transient events
We present the first multi-wavelength follow-up observations of two candidate
gravitational-wave (GW) transient events recorded by LIGO and Virgo in their
2009-2010 science run. The events were selected with low latency by the network
of GW detectors and their candidate sky locations were observed by the Swift
observatory. Image transient detection was used to analyze the collected
electromagnetic data, which were found to be consistent with background.
Off-line analysis of the GW data alone has also established that the selected
GW events show no evidence of an astrophysical origin; one of them is
consistent with background and the other one was a test, part of a "blind
injection challenge". With this work we demonstrate the feasibility of rapid
follow-ups of GW transients and establish the sensitivity improvement joint
electromagnetic and GW observations could bring. This is a first step toward an
electromagnetic follow-up program in the regime of routine detections with the
advanced GW instruments expected within this decade. In that regime
multi-wavelength observations will play a significant role in completing the
astrophysical identification of GW sources. We present the methods and results
from this first combined analysis and discuss its implications in terms of
sensitivity for the present and future instruments.Comment: Submitted for publication 2012 May 25, accepted 2012 October 25,
published 2012 November 21, in ApJS, 203, 28 (
http://stacks.iop.org/0067-0049/203/28 ); 14 pages, 3 figures, 6 tables;
LIGO-P1100038; Science summary at
http://www.ligo.org/science/Publication-S6LVSwift/index.php ; Public access
area to figures, tables at
https://dcc.ligo.org/cgi-bin/DocDB/ShowDocument?docid=p110003
A First Search for coincident Gravitational Waves and High Energy Neutrinos using LIGO, Virgo and ANTARES data from 2007
We present the results of the first search for gravitational wave bursts
associated with high energy neutrinos. Together, these messengers could reveal
new, hidden sources that are not observed by conventional photon astronomy,
particularly at high energy. Our search uses neutrinos detected by the
underwater neutrino telescope ANTARES in its 5 line configuration during the
period January - September 2007, which coincided with the fifth and first
science runs of LIGO and Virgo, respectively. The LIGO-Virgo data were analysed
for candidate gravitational-wave signals coincident in time and direction with
the neutrino events. No significant coincident events were observed. We place
limits on the density of joint high energy neutrino - gravitational wave
emission events in the local universe, and compare them with densities of
merger and core-collapse events.Comment: 19 pages, 8 figures, science summary page at
http://www.ligo.org/science/Publication-S5LV_ANTARES/index.php. Public access
area to figures, tables at
https://dcc.ligo.org/cgi-bin/DocDB/ShowDocument?docid=p120000
Search for gravitational waves associated with the InterPlanetary Network short gamma ray bursts
We outline the scientific motivation behind a search for gravitational waves
associated with short gamma ray bursts detected by the InterPlanetary Network
(IPN) during LIGO's fifth science run and Virgo's first science run. The IPN
localisation of short gamma ray bursts is limited to extended error boxes of
different shapes and sizes and a search on these error boxes poses a series of
challenges for data analysis. We will discuss these challenges and outline the
methods to optimise the search over these error boxes.Comment: Methods paper; Proceedings for Eduardo Amaldi 9 Conference on
Gravitational Waves, July 2011, Cardiff, U
First Low-Latency LIGO+Virgo Search for Binary Inspirals and their Electromagnetic Counterparts
Aims. The detection and measurement of gravitational-waves from coalescing
neutron-star binary systems is an important science goal for ground-based
gravitational-wave detectors. In addition to emitting gravitational-waves at
frequencies that span the most sensitive bands of the LIGO and Virgo detectors,
these sources are also amongst the most likely to produce an electromagnetic
counterpart to the gravitational-wave emission. A joint detection of the
gravitational-wave and electromagnetic signals would provide a powerful new
probe for astronomy.
Methods. During the period between September 19 and October 20, 2010, the
first low-latency search for gravitational-waves from binary inspirals in LIGO
and Virgo data was conducted. The resulting triggers were sent to
electromagnetic observatories for followup. We describe the generation and
processing of the low-latency gravitational-wave triggers. The results of the
electromagnetic image analysis will be described elsewhere.
Results. Over the course of the science run, three gravitational-wave
triggers passed all of the low-latency selection cuts. Of these, one was
followed up by several of our observational partners. Analysis of the
gravitational-wave data leads to an estimated false alarm rate of once every
6.4 days, falling far short of the requirement for a detection based solely on
gravitational-wave data.Comment: 13 pages, 13 figures. For a repository of data used in the
publication, go to:
http://dcc.ligo.org/cgi-bin/DocDB/ShowDocument?docid=P1100065 Also see the
announcement for this paper on ligo.org at:
http://www.ligo.org/science/Publication-S6CBCLowLatency
Should UI Eligibility Be Expanded to Low-Earning Workers? Evidence on Employment, Transfer Receipt, and Income from Administrative Data
Recent efforts to expand unemployment insurance (UI) eligibility are expected to increase low-earning workersâ access to UI. Although the expansionâs aim is to smooth the income and consumption of previously ineligible workers, it is possible that UI benefits simply displace other sources of income. Standard economic models predict that UI delays reemployment, thereby reducing wage income. Additionally, low-earning workers are often eligible for benefits from means-tested programs, which may decrease with UI benefits. In this paper, we estimate the impact of UI eligibility on employment, means-tested program participation, and income after job loss using a unique individual-level administrative data set from the state of Michigan. To identify a causal effect, we implement a fuzzy regression discontinuity design around the minimum earnings threshold for UI eligibility. Our main finding is that while UI eligibility increases jobless durations by up to 25 percent and temporarily lowers receipt of cash assistance (TANF) by 63 percent, the net impact on total income is still positive and large. In the quarter immediately following job loss, UI-eligible workers have 46-61 percent higher incomes than ineligibles
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