Top-down analysis of 30–80 kDa proteins by electron transfer dissociation time-of-flight mass spectrometry

Electron transfer dissociation (ETD)-based top-down mass spectrometry (MS) is the method of choice for in-depth structure characterization of large peptides, small- and medium-sized proteins, and non-covalent protein complexes. Here, we describe the performance of this approach for structural analysis of intact proteins as large as the 80 kDa serotransferrin. Current time-of-flight (TOF) MS technologies ensure adequate resolution and mass accuracy to simultaneously analyze intact 30-80 kDa protein ions and the complex mixture of their ETD product ions. Here, we show that ETD TOF MS is efficient and may provide extensive sequence information for unfolded and highly charged (around 1 charge/kDa) proteins of similar to 30 kDa and structural motifs embedded in larger proteins. Sequence regions protected by disulfide bonds within intact non-reduced proteins oftentimes remain uncharacterized due to the low efficiency of their fragmentation by ETD. For serotransferrin, reduction of S-S bonds leads to significantly varied ETD fragmentation pattern with higher sequence coverage of N- and C-terminal regions, providing a complementary structural information to top-down analysis of its oxidized form

Oxonium Ion-Guided Optimization of Ion Mobility-Assisted Glycoproteomics on the timsTOF Pro

Spatial separation of ions in the gas phase, providing information about their size as collisional cross-sections, can readily be achieved through ion mobility. The timsTOF Pro (Bruker Daltonics) series combines a trapped ion mobility device with a quadrupole, collision cell, and a time-of-flight analyzer to enable the analysis of ions at great speed. Here, we show that the timsTOF Pro is capable of physically separating N-glycopeptides from nonmodified peptides and producing high-quality fragmentation spectra, both beneficial for glycoproteomics analyses of complex samples. The glycan moieties enlarge the size of glycopeptides compared with nonmodified peptides, yielding a clear cluster in the mobilogram that, next to increased dynamic range from the physical separation of glycopeptides and nonmodified peptides, can be used to make an effective selection filter for directing the mass spectrometer to analytes of interest. We designed an approach where we (1) focused on a region of interest in the ion mobilogram and (2) applied stepped collision energies to obtain informative glycopeptide tandem mass spectra on the timsTOF Pro:glyco-polygon–stepped collision energy-parallel accumulation serial fragmentation. This method was applied to selected glycoproteins, human plasma– and neutrophil-derived glycopeptides. We show that the achieved physical separation in the region of interest allows for improved extraction of information from the samples, even at shorter liquid chromatography gradients of 15 min. We validated our approach on human neutrophil and plasma samples of known makeup, in which we captured the anticipated glycan heterogeneity (paucimannose, phosphomannose, high mannose, hybrid and complex glycans) from plasma and neutrophil samples at the expected abundances. As the method is compatible with off-the-shelve data acquisition routines and data analysis software, it can readily be applied by any laboratory with a timsTOF Pro and is reproducible as demonstrated by a comparison between two laboratories

Anti-Nogo-A Immunotherapy Does Not Alter Hippocampal Neurogenesis after Stroke in Adult Rats

Ischemic stroke is a leading cause of adult disability, including cognitive impairment. Our laboratory has previously shown that treatment with function-blocking antibodies against the neurite growth inhibitory protein Nogo-A promotes functional recovery after stroke in adult and aged rats, including enhancing spatial memory performance, for which the hippocampus is critically important. Since spatial memory has been linked to hippocampal neurogenesis, we investigated whether anti-Nogo-A treatment increases hippocampal neurogenesis after stroke. Adult rats were subject to permanent middle cerebral artery occlusion followed 1 week later by 2 weeks of antibody treatment. Cellular proliferation in the dentate gyrus was quantified at the end of treatment, and the number of newborn neurons was determined at 8 weeks post-stroke. Treatment with both anti-Nogo-A and control antibodies stimulated the accumulation of new microglia/macrophages in the dentate granule cell layer, but neither treatment increased cellular proliferation or the number of newborn neurons above stroke-only levels. These results suggest that anti-Nogo-A immunotherapy does not increase post-stroke hippocampal neurogenesis

Maxquant software for ion mobility enhanced shotgun proteomics

Ion mobility can add a dimension to LC-MS based shotgun proteomics which has the potential to boost proteome coverage, quantification accuracy and dynamic range. Required for this is suitable software that extracts the information contained in the four-dimensional (4D) data space spanned by m/z, retention time, ion mobility and signal intensity. Here we describe the ion mobility enhanced MaxQuant software, which utilizes the added data dimension. It offers an end to end computational workflow for the identification and quantification of peptides and proteins in LC-IMS-MS/MS shotgun proteomics data. We apply it to trapped ion mobility spectrometry (TIMS) coupled to a quadrupole time-of-flight (QTOF) analyzer. A highly parallelizable 4D feature detection algorithm extracts peaks which are assembled to isotope patterns. Masses are recalibrated with a non-linear m/z, retention time, ion mobility and signal intensity dependent model, based on peptides from the sample. A new matching between runs (MBR) algorithm that utilizes collisional cross section (CCS) values of MS1 features in the matching process significantly gains specificity from the extra dimension. Prerequisite for using CCS values in MBR is a relative alignment of the ion mobility values between the runs. The missing value problem in protein quantification over many samples is greatly reduced by CCS aware MBR.MS1 level label-free quantification is also implemented which proves to be highly precise and accurate on a benchmark dataset with known ground truth. MaxQuant for LC-IMS-MS/MS is part of the basic MaxQuant release and can be downloaded from http://maxquant.org

Parallel Accumulation–Serial Fragmentation (PASEF): Multiplying Sequencing Speed and Sensitivity by Synchronized Scans in a Trapped Ion Mobility Device

In liquid chromatography-mass spectrometry (LC-MS)-based proteomics, many precursors elute from the column simultaneously. In data-dependent analyses, these precursors are fragmented one at a time, whereas the others are discarded entirely. Here we employ trapped ion mobility spectrometry (TIMS) on an orthogonal quadrupole time-of-flight (QTOF) mass spectrometer to remove this limitation. In TIMS, all precursor ions are accumulated in parallel and released sequentially as a function of their ion mobility. Instead of selecting a single precursor mass with the quadrupole mass filter, we here implement synchronized scans in which the quadrupole is mass positioned with sub-millisecond switching times at the <i>m</i>/<i>z</i> values of appropriate precursors, such as those derived from a topN precursor list. We demonstrate serial selection and fragmentation of multiple precursors in single 50 ms TIMS scans. Parallel accumulation–serial fragmentation (PASEF) enables hundreds of MS/MS events per second at full sensitivity. Modeling the effect of such synchronized scans for shotgun proteomics, we estimate that about a 10-fold gain in sequencing speed should be achievable by PASEF without a decrease in sensitivity

Benefits of Collisional Cross Section Assisted Precursor Selection (caps-PASEF) for Cross-linking Mass Spectrometry

Ion mobility separates molecules in the gas-phase based on their physico-chemical properties, providing information about their size as collisional cross-sections. The timsTOF Pro combines trapped ion mobility with a quadrupole, collision cell and a time-of-flight mass analyzer, to probe ions at high speeds with on-the-fly fragmentation. Here, we show that on this platform ion mobility is beneficial for cross-linking mass spectrometry (XL-MS). Cross-linking reagents covalently link amino acids in close proximity, resulting in peptide pairs after proteolytic digestion. These cross-linked peptides are typically present at low abundance in the background of normal peptides, which can partially be resolved by using enrichable cross-linking reagents. Even with a very efficient enrichable cross-linking reagent, like PhoX, the analysis of cross-linked peptides is still hampered by the co-enrichment of peptides connected to a partially hydrolyzed reagent - termed mono-linked peptides. For experiments aiming to uncover protein-protein interactions these are unwanted byproducts. Here, we demonstrate that gas-phase separation by ion mobility enables the separation of mono-linked peptides from cross-linked peptide pairs. A clear partition between these two classes is observed at a CCS of 500 Å2 and a monoisotopic mass of 2 kDa, which can be used for targeted precursor selection. A total of 50 - 70% of the mono-linked peptides are prevented from sequencing, allowing the analysis to focus on sequencing the relevant cross-linked peptide pairs. In applications to both simple proteins and protein mixtures and a complete highly complex lysate this approach provides a substantial increase in detected cross-linked peptides

diaPASEF: parallel accumulation–serial fragmentation combined with data-independent acquisition

Data-independent acquisition modes isolate and concurrently fragment populations of different precursors by cycling through segments of a predefined precursor m/z range. Although these selection windows collectively cover the entire m/z range, overall, only a few per cent of all incoming ions are isolated for mass analysis. Here, we make use of the correlation of molecular weight and ion mobility in a trapped ion mobility device (timsTOF Pro) to devise a scan mode that samples up to 100% of the peptide precursor ion current in m/z and mobility windows. We extend an established targeted data extraction workflow by inclusion of the ion mobility dimension for both signal extraction and scoring and thereby increase the specificity for precursor identification. Data acquired from whole proteome digests and mixed organism samples demonstrate deep proteome coverage and a high degree of reproducibility as well as quantitative accuracy, even from 10 ng sample amounts.ISSN:1548-7105ISSN:1548-709