We are modern well equipped laboratory with the technical background able to perform most of the currently used UNTARGETED and TARGETED proteomic measurements. We provide full proteomics service, including protein isolation from the supplied sample to evaluation and consultation of the measured data. Since 2015, we handle approximately 300 orders per year (roughly 3000 samples). Results based on measurements from our laboratory were used in more than hundred of publications. To consult your plan, please contact Pavel Talacko (pavel.talacko@natur.cuni.cz) or Jana Jašprová (jana.jasprova@natur.cuni.cz).
Examples of analyses:
LFQ (label free quantification)
TMT (tandem mass tags)
TPP (Thermal proteome profiling)
phosphoproteome profiling
pull-down experiments
A basic type of proteomic experiment. Complete lysate is digested with trypsin. The resulting peptides are desalted and separated on a reverse phase column. Due to the low amount of proteins and the high complexity of the sample, liquid chromatography is currently most often used in nanoflow - hundreds of nanoliters per minute. Liquid chromatography (Vanquish Neo) is directly connected to the electrospray of the Orbitrap Fusion, Orbitrap Ascend or Orbitrap Astral mass spectrometer.
The mass spectrometer measures alternately in two modes: MS1, where peptide masses are measured with high resolution and accuracy, and MS2, in which peptides selected from the previous MS1 scan are fragmented.
Accurate quantification of selected proteins.
Method aims to validate the results from an untargeted approach. It is performed on an Orbitrap using MRM method. It is an alternative approach to common techniques such as western blot. The advantage of this approach is the possibility to analyze several proteins (dozens) at once during one injection. This eliminates the problem of finding suitable antibodies.
Parallel analysis of peptides labeled with isotopically labeled (heavy) amino acids is essential. These serve as an internal standards.
The amount of proteins that can be targeted in one injection is strongly dependent on the concentration of the target proteins in the sample. The lower the expression of the target proteins, the less it is possible to analyze them in one injection due to the need for higher loading times. The information of fragmentation properties of targeted peptides is important and is obtained from an untargeted experiment. However, in some cases fragmentation information can be obtained from publicly available databases.
Tips for sample handling:
do not use trypsin for cell harvesting
inform us about presence of detergent
always keep in mind same protein loading for pull-down experiments (eg. Co-immunoprecipitation)
do not use the phosphatase inhibitors for phosphoproteomic experiments