Instrumentation

1. Mass Spectrometry

The FZI Core Facility Proteomics is equipped with four state-of-the-art UPLC-MS platforms for highly sensitive and precise quantitative proteomics studies allowing high-throughput analyses of cohorts of hundreds of samples. This includes three timsTOF Pro-2 (Bruker Daltonik) instruments coupled to nanoElute, Evosep One and nanoAcquity LCs; and one Orbitrap Exploris 480 platforms with FAIMS Pro interface, coupled to an Ultimate 3000 RSLCnano system (Thermo Fischer Scientific).

 

The timsTOF Pro platforms offer sample acquisition at high resolution, fast scanning speed, enhanced specificity and high sensitivity. When coupled to mass spectrometry (MS), ion mobility spectrometry (IMS) allows the separation of ions based on their structure and mass-to-charge ratios. By trapped ion mobility spectrometry (TIMS) a fourth dimension is added, as ions can also be separated according to their mobility. The Bruker timsTOF Pro instruments offer online parallel accumulation-serial fragmentation (PASEF) – a recently introduced scan mode allowing faster sequencing rates without losing sensitivity. Taken together, these instrument platforms allow ion separation with high efficiency, duty cycle, at a high resolving power and are highly suitable for analyses of larger sample cohorts.

 

The Orbitrap Exploris 480 mass spectrometery platform (Thermo Fisher Scientific) is equipped with an electric field asymmetric ion mobility spectroscopy (FAIMS) interface that reduces time consuming sample processing steps and matrix effects and improves proteome coverage. These features are especially relevant in experiments with limited sample amounts or in single-cell proteomics – but is of course not limited to these experimental settings. By combining the FAIMS Pro interface and data-independent acquisition (DIA) strategies analyses times can be significantly reduced due to the application of shorter gradients which are especially suited for high sample throughput.

 

 

 

For high-throughput sample preparation a Biomek i7 automated work station is available in our lab. This offers the opportunity of highly standardized sample processing procedures including FASP- and SP3-based protocols.

 

 

 

 

 


2. Protein Purification

 

GE Äkta Pure 25 Purification System

 

The GE Äkta Pure 25 Purification System serves as versatile chromatography platform for the purification and separation of proteins and peptides. Similar to High Performance Liquid Chromatography (HPLC), the Äkta Fast Protein Liquid Chromatography (FPLC) workflow usually consists of injection, separation, detection and fractionation.

 

 

 

 

 

Our lab offers flexible use of different modules in each part of the workflow:

Injection

The injectable volumes range between 1 µL and 2000 µL. Using the Äkta Sample Pump or the manual injection port, fixed volumes of 100 µL, 200 µL, 500 µL, 1000 µL and 2000 µL can be easily manually injected. Our SparkLine Alias Autosampler allows the automated injection of variable volumes from 1 µL to 1000 µL for up to 96 samples. Furthermore, a SuperLoop can be used to apply varying amounts of sample directly onto the separation column.

Separation

By using different stationary phases, multiple analytical and/or preparative tasks can be solved. Frequently used methods include Size Exclusion Chromatography (SEC) and affinity chromatography for protein purification as well as Electrostatic Repulsion Chromatography (ERLIC) for phosphopeptide enrichment. This system offers a binary pump with up to seven different inlets for elution medium B and two fixed inlets for elution medium A.

 

Detection

By default we rely UV/VIS detection at typically 280 nm and 214 nm followed by conductivity detection. Additionally, Äkta Pure 25 offers the possibility of pH-monitoring in aqueous media.

 

Fractionation

The fraction collector allows the continuous or signal triggered collection of eluting samples. By using modern in-house 3D printing technology, we were able to create custom built tube holders for the flexible use of low-binding tubes and plates for sample collection. Currently available collection systems are e.g. common 96well deepwell plates, 15 mL and 50 mL Falcon tubes, 5 mL and 1.5 mL Eppendorf tubes and 200 µL PCR tubes.

 

For further performance specification, please see ÄKTA™ pure User Manual or contact us.

 


Dionex UltiMate 3000

The Dionex Ultimate 3000 is our High Performance Liquid Chromatography (HPLC) system for versatile solutions of several analytical problems. In contrast to a classical FPLC, this HPLC system can produce and withstand higher pressures and therefore allows to use smaller particle sizes and column diameters. The system is fully biocompatible and provides high resolution separation of small sample quantities in short analysis times. This is especially important when the sample quantity is limited, e.g. for offline fractionation of proteomic samples. Currently applied methods include Immobilized Metal-Ion Affinity Chromatography for phosphopeptide enrichment (IMAC), high-pH reversed phase, Strong Cation Exchange (SCX) and Strong Anion Exchange (SAX) peptide fractionation and small molecule analysis. Although the separation of larger preparative sample sizes is possible in principle, the system is currently equipped with modules optimized for analytical scale separations. For larger sample sizes and preparative tasks (such as protein purification by affinity chromatography), the Äkta Pure FPLC system is the system of choice.

 

Injection

The injectable volumes can be adjusted to every available loop size. Usually our system is equipped with injection loops of analytical size. The injection mode is variable (e.g. full loop, partial loop etc.) and automated by the autosampler.

 

Separation

We offer a range of common analytical and separation columns for peptide and small molecule analyses including Metal-Ion loaded NTA columns, SAX/WAX and reversed phase material in multiple scales (i.e. particle sizes, column diameters and column lengths). Flow rates of up to 8 ml/min allow high pressures (up to 100 MPa) within the system in order to provide more flexibility with respect to column formats and particle sizes.

 

Detection

By default, we offer UV/VIS detection by two different detectors. The VWD RS detector is equipped with a regular size flow cell (180 nl). It allows monitoring of analytes with different wavelengths in up to four different UV/VIS channels at 5 Hz (single wavelength 200 Hz). In addition, our system is equipped with a second VWD harbouring a micro-volume flow cell (3 nL).

 

Fractionation

The fraction collector allows the continuous or signal triggered collection of eluting samples in different tube formats.

 

For further information, please visit the ThermoFisher Homepage