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 “on-the-fly”. 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 protein–protein 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 CL–MS 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 MS¹ 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 CL–MS that is useful for any sample where SILAC labeling may not be practical

Indirect calorimetry of FAAH^−/− 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 −15 h, day re-fed −5 h in duration. n = 8, 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^−/− mice.</p

FAAH deficiency causes hepatic and adipose insulin resistance.

<p>Glycerol production, and hepatic glucose production from glycerol, assessed using a [2-¹³C] 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. n = 5, data are mean ± SEM. *p<0.05, ***p<0.001 wild-type vs. FAAH^−/− mice by Student's t-test.</p

Plasma glucose and insulin during [U-¹³C₆] glucose pump assessment following an 18 hr fast.

<p>Data are mean ± SEM.</p>***<p>P<0.001, wild-type vs. FAAH^−/− mice by Student's t-test.</p