Proteomics services

Description: this type of analysis is performed on SDS PAGE (either denaturing or native) as well as other type of gel electrophoresis platforms. The main goal of this bottom-up analysis is to identify the identity of an electrophoretically separated gel band by destaining it and digest the protein material in it. The resulting peptides are collected, cleaned-up and injected into the system. The collected spectra are used to search a relevant protein database and provide the identity of all detected proteins. 

Amount needed: a general rule of thumb is that a gel band visible to the naked eye will produce enough signal for identification.  In general, amount of at least 100 ng are needed for a good signal.

Type of samples: any protein that can be electrohoretically separated on an SDS-PAGE

 MSF protocol available: yes

Degree of difficulty: low

LC gradient length: up to 2 hours, usually < 1 hour.

Notes: please note that some staining agents such as Silver stain are not compatible with this type of analysis. If you are not sure about the compatibility of your electrohoretic system with mass spec, contact the facility. 
Also, please note the western blotting is not compatible with this type of analysis, although more complex sample prep techniques may produce enough signal.

Description: the goal of this type of analysis is to identify and quantify in a relative manner as many proteins as possible between different sample groups. Samples are processed all together using the same amount of starting material (when possible). The resulting peptides are quantified spectrophotometrically, and the same amount is injected on the instrument. MS/MS spectra are used for identification, while the area under the curve of extracted ion chromatograms is used for the quantification. This approach is preferred when the user needs to compare many conditions among each other. For example, a user may want to compare the effect of a drug over a time using a series of time points. In this case, a label-free approach provides a cost-effective compromise to obtain high quality data that inform future experiments.

Amount needed: a minimum of 50 µg is desirable but can be performed on amount as little as 10 µg. Smaller amount may still provide enough signal, but sample loss during sample prep becomes a potential issue.

Type of samples: bacterial cells and biofilms, plant or animal tissue and cells.

MSF protocol available: yes

Degree of difficulty: medium to high, depending on the type of sample.

Gradient length: up to 2 hours per samples/fraction

Notes: label free quantification relies on the analysis of samples one after the other. This may lead to sample queue effects (e.g., decreased response over time) especially for long (> 24 hours) sequences. The MSF randomizes injections to provide an unbiased result, but it is advisable to increase the number of replicates from 3 to 4.

Description: the goal of this type of analysis is to identify and quantify in a relative manner as many proteins as possible between different sample groups. Samples are processed all together using the same amount of starting material (when possible). The resulting peptides are quantified spectrophotometrically and tagged with an isobaric reagent (TMT tabs) that adds the same identical mass to each peptide with each reaction. The same number of peptides are then combined in a pooled samples that is then fractionated or injected into the system. The intensity of each peptide is the result of the combined contribution of each sample to the pool. Upon fragmentation, each TMT tag releases a reporter ion that as a unique mass associated with its sample. The intensity of these reporter ions is used to determine the ratio of that specific peptides in each sample. Since the peptide present in each sample is analyzed at the same exact time, the analysis does not suffer from the variability affecting label-free experiments. Currently, the MSF is using a six-plex (six tags) as the preferred method. This allows comparison of 6 samples, usually a triplicate control vs, a triplicate condition. TMT tagging is the preferred approach when the user needs to compare two samples with highest possible precision because the majority of the quantified proteins has coefficient of variabilities < 20%.  

Amount needed: a minimum of 50 µg is desirable but can be performed on amount as little as 20 µg. Smaller amount may still provide enough signal, but sample loss during sample prep becomes a potential issue.

Type of samples: bacterial cells and biofilms, plant or animal tissue and cells.

MSF protocol available: yes

Degree of difficulty: medium to high, depending on the type of sample.

Gradient length: up to 2 hours per samples/fraction

Notes: TMT tags are expensive. Moreover, these experiment offer require fractionation to fully explore the proteome. Therefore, this approach is preferred when two conditions needs to be compare. If the user wants to compare the same control with multiple conditions, each comparison will require tagging and a separate set of runs.

Description: the goal of this type of analysis is to quantify a set of proteins using 2 or more peptides. On the MSF Q-Exactive platform, this goal is achieved through a process called parallel reaction monitoring (PRM). Precursor ions are isolated in the quadrupole and accumulated in the C-trap before being fragmented in the high energy collision cell. The resulting fragments are used to verify the identity of the peptide, and the most intense fragments are used to produce an extracted ion chromatogram that is then integrated. This process can be performed in a scheduled manner, which enables detection of hundreds of parent ions in a single run. A common approach to PRM analysis relies on analysis of a QC pool sample in untargeted mode, which allows the creation of a spectral library database as well as a reference of retention time and spectra for the peptides of interest. Afterward, samples are run sequentially using a schedule based on the QC sample. This approach is often used to verify the relative quantification of a TMT or label-free experiment for a subset of proteins of interest detected in previous experiments.

Amount needed: targeted method may require variable amounts depending on the abundance of the target protein(s). The loading limit on our columns is 2 µg of tryptic peptide, and this amount can be achieved with as little as 5 µg of starting material.

Type of samples: bacterial cells and biofilms, plant or animal tissue and cells.

MSF protocol available: yes (any protocol for untargeted proteomics works)

Degree of difficulty: medium to high, depending on the type of sample.

Gradient length: up to 2 hours per samples/fraction, often < 1 hour

Notes: targeted methods for absolute quantification (i.e. m/v) require a lengthy development and validation process and the purchase of labelled peptides for internal standard and quantification. Users considering a targeted method may want to keep the leftover peptides of an untargeted method, for they may be used for targeted experiments.

Description: the goal of this type of analysis is to identify and quantify protein modifications that happened after the protein were translated by the ribosomes or after certain event occurred within a cell or tissue. Some of the most common glycosylation, phosphorylation, ubiquitination, acylation, methylation, and acetylation, and their occurrence may vary from minimal (<1% of the protein pool) to complete (100% of the protein pool). PTMs often result in multiple versions of the same peptide, one modified and one note, which is a direct consequence of the presence of modified and unmodified proteins in the matrix under analysis. It is quite common for PTMs to require specific sample preparation step that ensure the integrity of the modification as well as the enrichment of proteins carrying it. PTMs analysis can be carried out in label-free, labelled and targeted fashion. It is also possible to search for known PTMs in dataset that have been previously collected.

Amount needed: depends on the PTM. For example, phosphorylation will require ~ 10x more starting material and an enrichment step to provide good signal for a significant number of peptides.

Type of samples: bacterial cells and biofilms, plant or animal tissue and cells.

MSF protocol available: for some PTMs

Degree of difficulty: generally high, depending on the type of PTM.

Gradient length: up to 2 hours per sample/fraction

Notes: PTMs analysis is almost always sensitive to sample prep at a rate much higher than normal proteomics experiments. Therefore, particular emphasis must be placed on protocol validation to ensure a successful execution of these experiments.

Description: the goal of this type of analysis is to identify interactions between proteins or between a protein and a target (an agonist, an antagonist etc.). Interaction between a protein and other targets can be investigated by crosslinking a sample that contains them. In this case, the resulting sample may show peptides that have crosslinks among them, showing their origin, and targeting the crosslinker through a neutral loss analysis may allow targeted acquisition of these peptides. Another approach utilizes an enrichment strategy based on conjugation of the protein to be tested with magnetic beads, which are then mix with a protein lysate. After washing of the beads and protein elution, the resulting sample contains the target proteins and all the proteins that interacted with it.

Amount needed: depends on the experiment. In the case of enrichment experiments, our general advice is to run an SDS gel and verify that there are potential bands of interest. For crosslinking experiments, we suggest running a single sample on our platform to verify detection of crosslinked peptides.

Type of samples: bacterial cells and biofilms, plant or animal tissue and cells.

MSF protocol available: no. These protocols are very specific to the type of experiment. The MSF may offer suggestions on integration of existing protocol with mass spectrometry.

Degree of difficulty: generally high, mostly because of the low amount of final sample.

Gradient length: up to 2 hours per sample/fraction

Notes: these experiments are often adapted from existing protocols for Western blotting or other single protein detection systems. It is highly advised to contact the MSF and enquire about the adaptability of these protocols to our platforms.

Description: the goal of this analysis is to detect the signal of intact proteins, without the need for an enzymatic digestion step. This goal can be achieved in denaturing or native conditions. In denaturing condition, the protein is exchanged to a buffer or solvent compatible with mass spec (often water with 0.1% formic acid or a 10 mM ammonium bicarbonate buffer), and the runs are performed with solvent containing acids (e.g., 0.1% formic acid). Proteins analyzed in these conditions are often well unfolded and provide signals with extensive multiple charging. In native conditions, both the samples and the HPLC solvents are kept at neutral pH using buffers, which keeps the proteins partially or almost completely folded. This results in signals with fewer charges, which can be used to interrogate the actual structure of the protein.

Very often these analyses are performed simply to get the molecular weight of a protein of interest (e.g., after expression and purification). There are also cases in which the analysis is performed to check if a protein is interacting with a ligand, or with a metal cluster.  This analysis can be performed with a column, in flow-through or in infusion mode.

Amount needed: in denaturing condition, solution with concentration as low as 1 µM may provide enough signal. In native conditions, it is advisable to increase the concentration up to 50 folds.

Type of samples: any purified protein. The MSF does not analyze protein mixtures in this mode.

MSF protocol available: we have protocols available for buffer exchange.

Degree of difficulty: generally low.

Gradient length: if a column is used, the gradient is usually 15 minutes.

Notes: while we have the ability to perform online desalting, the type of salts and concentrations used in biology labs are often too high for our system. Therefore, we strongly suggest performing a buffer exchange step.