36 research outputs found
Parallel Spectral Acquisition with an Ion Cyclotron Resonance Cell Array
Mass measurement accuracy is a critical
analytical figure-of-merit
in most areas of mass spectrometry application. However, the time
required for acquisition of high-resolution, high mass accuracy data
limits many applications and is an aspect under continual pressure
for development. Current efforts target implementation of higher electrostatic
and magnetic fields because ion oscillatory frequencies increase linearly
with field strength. As such, the time required for spectral acquisition
of a given resolving power and mass accuracy decreases linearly with
increasing fields. Mass spectrometer developments to include multiple
high-resolution detectors that can be operated in parallel could further
decrease the acquisition time by a factor of <i>n</i>, the
number of detectors. Efforts described here resulted in development
of an instrument with a set of Fourier transform ion cyclotron resonance
(ICR) cells as detectors that constitute the first MS array capable
of parallel high-resolution spectral acquisition. ICR cell array systems
consisting of three or five cells were constructed with printed circuit
boards and installed within a single superconducting magnet and vacuum
system. Independent ion populations were injected and trapped within
each cell in the array. Upon filling the array, all ions in all cells
were simultaneously excited and ICR signals from each cell were independently
amplified and recorded in parallel. Presented here are the initial
results of successful parallel spectral acquisition, parallel mass
spectrometry (MS) and MS/MS measurements, and parallel high-resolution
acquisition with the MS array system
Accurate Peptide Fragment Mass Analysis: Multiplexed Peptide Identification and Quantification
Fourier transform-all reaction monitoring (FT-ARM) is
a novel approach
for the identification and quantification of peptides that relies
upon the selectivity of high mass accuracy data and the specificity
of peptide fragmentation patterns. An FT-ARM experiment involves continuous,
data-independent, high mass accuracy MS/MS acquisition spanning a
defined <i>m</i>/<i>z</i> range. Custom software
was developed to search peptides against the multiplexed fragmentation
spectra by comparing theoretical or empirical fragment ions against
every fragmentation spectrum across the entire acquisition. A dot
product score is calculated against each spectrum to generate a score
chromatogram used for both identification and quantification. Chromatographic
elution profile characteristics are not used to cluster precursor
peptide signals to their respective fragment ions. FT-ARM identifications
are demonstrated to be complementary to conventional data-dependent
shotgun analysis, especially in cases where the data-dependent method
fails because of fragmenting multiple overlapping precursors. The
sensitivity, robustness, and specificity of FT-ARM quantification
are shown to be analogous to selected reaction monitoring-based peptide
quantification with the added benefit of minimal assay development.
Thus, FT-ARM is demonstrated to be a novel and complementary data
acquisition, identification, and quantification method for the large
scale analysis of peptides
Mango: A General Tool for Collision Induced Dissociation-Cleavable Cross-Linked Peptide Identification
Chemical cross-linking combined with
mass spectrometry provides
a method to study protein structures and interactions. The introduction
of cleavable bonds in a cross-linker provides an avenue to decouple
released peptide masses from their precursor species, greatly simplifying
the downstream search, allowing for whole proteome investigations
to be performed. Typically, these experiments have been challenging
to carry out, often utilizing nonstandard methods to fully identify
cross-linked peptides. Mango is an open source software tool that
extracts precursor masses from chimeric spectra generated using cleavable
cross-linkers, greatly simplifying the downstream search. As it is
designed to work with chimeric spectra, Mango can be used on traditional
high-resolution tandem mass spectrometry (MS/MS) capable mass spectrometers
without the need for additional modifications. When paired with a
traditional proteomics search engine, Mango can be used to identify
several thousand cross-linked peptide pairs searching against the
entire <i>Escherichia coli</i> proteome. Mango provides
an avenue to perform whole proteome cross-linking experiments without
specialized instrumentation or access to nonstandard methods
Mango: A General Tool for Collision Induced Dissociation-Cleavable Cross-Linked Peptide Identification
Chemical cross-linking combined with
mass spectrometry provides
a method to study protein structures and interactions. The introduction
of cleavable bonds in a cross-linker provides an avenue to decouple
released peptide masses from their precursor species, greatly simplifying
the downstream search, allowing for whole proteome investigations
to be performed. Typically, these experiments have been challenging
to carry out, often utilizing nonstandard methods to fully identify
cross-linked peptides. Mango is an open source software tool that
extracts precursor masses from chimeric spectra generated using cleavable
cross-linkers, greatly simplifying the downstream search. As it is
designed to work with chimeric spectra, Mango can be used on traditional
high-resolution tandem mass spectrometry (MS/MS) capable mass spectrometers
without the need for additional modifications. When paired with a
traditional proteomics search engine, Mango can be used to identify
several thousand cross-linked peptide pairs searching against the
entire <i>Escherichia coli</i> proteome. Mango provides
an avenue to perform whole proteome cross-linking experiments without
specialized instrumentation or access to nonstandard methods
<i>In Vivo</i> Protein Interaction Network Identified with a Novel Real-Time Cross-Linked Peptide Identification Strategy
Protein interaction topologies are
critical determinants of biological
function. Large-scale or proteome-wide measurements of protein interaction
topologies in cells currently pose an unmet challenge that could dramatically
improve understanding of complex biological systems. A primary impediment
includes direct protein topology and interaction measurements from
living systems since interactions that lack biological significance
may be introduced during cell lysis. Furthermore, many biologically
relevant protein interactions will likely not survive the lysis/sample
preparation and may only be measured with <i>in vivo</i> methods. As a step toward meeting this challenge, a new mass spectrometry
method called <b>Re</b>al-time <b>A</b>nalysis for <b>C</b>ross-linked peptide <b>T</b>echnology (ReACT) has been
developed that enables assignment of cross-linked peptides âon-the-flyâ.
Using ReACT, 708 unique cross-linked (<5% FDR) peptide pairs were
identified from cross-linked <i>E. coli</i> cells. These
data allow assembly of the first protein interaction network that
also contains topological features of every interaction, as it existed
in cells during cross-linker application. Of the identified interprotein
cross-linked peptide pairs, 40% are derived from known interactions
and provide new topological data that can help visualize how these
interactions exist in cells. Other identified cross-linked peptide
pairs are from proteins known to be involved within the same complex,
but yield newly discovered direct physical interactors. ReACT enables
the first view of these interactions inside cells, and the results
acquired with this method suggest cross-linking can play a major role
in future efforts to map the interactome in cells
Large-Scale and Targeted Quantitative Cross-Linking MS Using Isotope-Labeled Protein Interaction Reporter (PIR) Cross-Linkers
Quantitative
measurement of chemically cross-linked proteins is
crucial to reveal dynamic information about protein structures and
proteinâprotein interactions and how these are differentially
regulated during stress, aging, drug treatment, and most perturbations.
Previously, we demonstrated how quantitative in vivo cross-linking
(CL) with stable isotope labeling of amino acids in cell culture (SILAC)
enables both heritable and dynamic changes in cells to be visualized.
In this work, we demonstrate the technical feasibility of proteome-scale
quantitative in vivo CLâMS using isotope-labeled protein interaction
reporter (PIR) cross-linkers and <i>E. coli</i> as a model
system. This isotope-labeled cross-linkers approach, combined with
Real-time Analysis of Cross-linked peptide Technology (ReACT) previously
developed in our lab, enables the quantification of 941 nonredundant
cross-linked peptide pairs from a total of 1213 fully identified peptide
pairs in two biological replicate samples through comparison of MS<sup>1</sup> peak intensity of the light and heavy cross-linked peptide
pairs. For targeted relative quantification of cross-linked peptide
pairs, we further developed a PRM-based assay to accurately probe
specific site interaction changes in a complex background. The methodology
described in this work provides reliable tools for both large-scale
and targeted quantitative CLâMS that is useful for any sample
where SILAC labeling may not be practical
Indirect calorimetry of FAAH<sup>â/â</sup> and wild-type mice.
<p>A) hr-hr B) 12 hr average. a) Oxygen consumption (VO2), b) Respiratory exchange ratio (RER) and c) Activity during the diurnal cycle and fasted to fed transitions. Day (light cycle) and night (dark cycle) 12 hours, (over) night fast â15 h, day re-fed â5 h in duration. nâ=â8, data are mean ± SEM, *p<0.05, **p<0.01 by Student's t-test.</p
General body composition, basal glucose and insulin.
<p>Data are mean±SEM.</p>*<p>p<0.05,</p>**<p>p<0.01 by Student's t-test for wild-type vs. FAAH<sup>â/â</sup> mice.</p
FAAH deficiency causes hepatic and adipose insulin resistance.
<p>Glycerol production, and hepatic glucose production from glycerol, assessed using a [2-<sup>13</sup>C] glycerol infusion administered by Alza miniosmotic pump. Glycerol production represents mainly in vivo lipolysis, and was measured after 18 h of overnight fast. Fasted plasma glucose and insulin levels are given in the table. Glycerol production rate is expressed in terms of mg produced/kg of body weight/minute. nâ=â5, data are mean ± SEM. *p<0.05, ***p<0.001 wild-type vs. FAAH<sup>â/â</sup> mice by Student's t-test.</p
Plasma glucose and insulin during [U-<sup>13</sup>C<sub>6</sub>] glucose pump assessment following an 18 hr fast.
<p>Data are mean ± SEM.</p>***<p>P<0.001, wild-type vs. FAAH<sup>â/â</sup> mice by Student's t-test.</p