Biology subjects

Meier, F.

Publications and source records attributed to Meier, F..

5 recordsLinked to original sources

bioRxiv2018

MaxQuant.Live enables global targeting of more than 25,000 peptides

Mass spectrometry (MS)-based proteomics is often performed in a shotgun format, in which as many peptide precursors as possible are selected from full or MS1 scans so that their fragment spectra can be recorded in MS2 scans. While achieving great proteome depths, shotgun proteomics cannot guarantee that each precursor will be fragmented in each run. In contrast, targeted proteomics aims to reproducibly and sensitively record a restricted number of precursor/fragment combinations in each run, based on pre-scheduled mass-to-charge and retention time windows. Here we set out to merge these two concepts by a global targeting approach in which an arbitrary number of precursors of interest are detected in real-time, followed by standard fragmentation or advanced peptide-specific analyses. We made use of a fast application programming interface to a quadrupole Orbitrap instrument and real-time recalibration in mass, retention time and intensity dimensions to predict precursor identity. MaxQuant.Live is freely available (www.maxquant.live) and has a graphical user interface to specify many pre-defined data acquisition strategies. Acquisition speed is as fast as with the vendor software and the power of our approach is demonstrated with the acquisition of breakdown curves for hundreds of precursors of interest. We also uncover precursors that are not even visible in MS1 scans, using elution time prediction based on the auto-adjusted retention time alone. Finally, we successfully recognized and targeted more than 25,000 peptides in single LC-MS runs. Global targeting combines the advantages of two classical approaches in MS-based proteomics, while greatly expanding the analytical toolbox.

bioinformatics↗

bioRxiv2018

A hippocampal model for behavioral time acquisition and fast bidirectional replay of spatio-temporal memory sequences

The hippocampus is known to play a crucial role in the formation of long-term memory. For this, fast replays of previously experienced activities during sleep or after reward experiences are believed to be crucial. But how such replays are generated is still completely unclear. In this paper we propose a possible mechanism for this: we present a model that can store experienced trajectories on a behavioral timescale after a single run, and can subsequently bidirectionally replay such trajectories, thereby omitting any specifics of the previous behavior like speed, etc, but allowing repetitions of events, even with different subsequent events. Our solution builds on well-known concepts, one-shot learning and synfire chains, enhancing them by additional mechanisms using global inhibition and disinhibition. For replays our approach relies on dendritic spikes and cholinergic modulation, as supported by experimental data. We also hypothesize a functional role of disinhibition as a pacemaker during behavioral time.

neuroscience↗

bioRxiv2018

Online parallel accumulation - serial fragmentation (PASEF) with a novel trapped ion mobility mass spectrometer

In bottom-up proteomics, peptides are separated by liquid chromatography with elution peak widths in the range of seconds, while mass spectra are acquired in about 100 microseconds with time-of-fight (TOF) instruments. This allows adding ion mobility as a third dimension of separation. Among several formats, trapped ion mobility spectrometry (TIMS) is attractive due to its small size, low voltage requirements and high efficiency of ion utilization. We have recently demonstrated a scan mode termed parallel accumulation - serial fragmentation (PASEF), which multiplies the sequencing speed without any loss in sensitivity (Meier et al., PMID: 26538118). Here we introduce the timsTOF Pro instrument, which optimally implements online PASEF. It features an orthogonal ion path into the ion mobility device, limiting the amount of debris entering the instrument and making it very robust in daily operation. We investigate different precursor selection schemes for shotgun proteomics to optimally allocate in excess of 100 fragmentation events per second. More than 800,000 fragmentation spectra in standard 120 min LC runs are easily achievable, which can be used for near exhaustive precursor selection in complex mixtures or re-sequencing weak precursors. MaxQuant identified more than 6,400 proteins in single run HeLa analyses without matching to a library, and with high quantitative reproducibility (R > 0.97). Online PASEF achieves a remarkable sensitivity with more than 2,900 proteins identified in 30 min runs of only 10 ng HeLa digest. We also show that highly reproducible collisional cross sections can be acquired on a large scale (R > 0.99). PASEF on the timsTOF Pro is a valuable addition to the technological toolbox in proteomics, with a number of unique operating modes that are only beginning to be explored.

systems biology↗

bioRxiv2018

A novel LC system embeds analytes in pre-formed gradients for rapid, ultra-robust proteomics

To further integrate mass spectrometry (MS)-based proteomics into biomedical research and especially into clinical settings, high throughput and robustness are essential requirements. They are largely met in high-flow rate chromatographic systems for small molecules but these are not sufficiently sensitive for proteomics applications. Here we describe a new concept that delivers on these requirements while maintaining the sensitivity of current nano-flow LC systems. Low-pressure pumps elute the sample from a disposable trap column, simultaneously forming a chromatographic gradient that is stored in a long storage loop. An auxiliary gradient creates an offset, ensuring the re-focusing of the peptides before the separation on the analytical column by a single high-pressure pump. This simplified design enables robust operation over thousands of sample injections. Furthermore, the steps between injections are performed in parallel, reducing overhead time to a few minutes and allowing analysis of more than 200 samples per day. From fractionated HeLa cell lysates, deep proteomes covering more than 130,000 sequence unique peptides and close to 10,000 proteins were rapidly acquired. Using this data as a library, we demonstrate quantitation of 5200 proteins in only 21 min. Thus, the new system-termed Evosep One-analyzes samples in an extremely robust and high throughput manner, without sacrificing in depth proteomics coverage.

biochemistry↗

bioRxiv2017

EASI-tag enables accurate multiplexed and interference-free MS2-based proteome quantification

We developed EASI-tag (Easily Abstractable Sulfoxide-based Isobaric tag), a new generation of amine-derivatizing and sulfoxide-containing isobaric labelling reagents, which dissociate at low collision energy and generate peptide-coupled, interference-free reporter ions with high yield. Efficient isolation of 12C precursors and quantification at the MS2 level enable accurate determination of quantitative differences between multiplexed samples. EASI-tag makes isobaric labeling applicable to any bottom up proteomics workflow and to benchtop mass spectrometers.

biochemistry↗