Identification of proteins in complex mixtures
Identification of protein modifications
Identification of proteins from gel bands
Custom protocols
Identification of proteins in complex mixtures top
Nanoscale LC MS/MS allows the identification of individual peptides from a complex mixture. This is a powerful proteomics technique through which the component proteins in a sample can be identified.
Typically, a sample is prepared by tryptic digestion and desalting of a protein mixture obtained in the course of the users’ research. Samples have varied in complexity from purified protein complexes to whole cell extracts.
We load desalted, proteolysed mixture on a nanoscale HPLC column. These columns employ either reverse phase (1-dimensional) or a combination of ion exchange and reverse phase (2-dimensional) separation chemistries. The eluent from the LC column is subjected directly to tandem mass spectroscopy, and the mass and fragmentation spectrum of major ions is recorded.
A computer program, Sequest, is then used to identify the peptides that gave the spectra that have been collected. It queries a sequence database for the appropriate organism, calculating theoretical fragmentation spectra for all possible peptides and comparing them to the data collected.
The final output for the user is a file listing each gene for which peptides were found in the data. Each peptide is listed along with statistics showing the quality of the data.
One dimensional separations are appropriate for samples where the expected complexity is 1 to 10 proteins. Between 10 ng and 500 ng are needed for the analysis.
Two dimensional “MudPit” separations as developed by the Yates lab are appropriate for more complex mixtures. Between 1 ug and 100 ug are needed for analysis.
Three dimensional separations employ a three phase nano LC column. In this technique samples are desalted after loading the nano LC column. Three dimensional separations are appropriate for mixtures containing peptides that do not bind well to reverse phase media and for situations where very high sequence coverage or high performance identification is necessary.
Users generally prepare a proteolysed, desalted sample for one dimensional or two dimensional analysis. For three dimensional analysis, the sample is prepared by digestion but is not desalted.
To perform this type of analysis, please refer to the following forms and protocols:
Enzymatic digestion of protein samples
Sample desalting for mass spectrometry
Sample submission form
We urge you to contact facility staff to discuss your project prior to sample preparation.
Identification of protein modifications top
LC MS/MS data can be processed to detect any modifications that may be present in the peptides detected. The modification must add a calculatable molecular weight. Modification analysis can be performed using either one dimensional or multidimensional “MudPit” chromatography depending on the sample complexity. We can often find modifications on a target protein even when it is part of a complex mixture. Often modifications of interest can be found simply by searching LC mass spec data collected under standard conditions. For some types of modification including phosphorylation, we can collect additional data that confirms the presence of the modification with higher confidence. Ask us for details.
In some cases, chances of pinpointing the site of a modification increase if overlapping peptides are analyzed. To produce sets of overlapping peptides, we recommend digestion with two or three proteases that vary in their specificity.
If there is a suspected site of modification, the proteolytic digest can be planned to produce peptides of optimal mass containing that site. You can use peptidecutter to choose an appropriate protease.
To perform this type of analysis, please refer to the following forms and protocols:
Enzymatic digestion of protein samples
Triple enzymatic digestion of protein samples
Sample desalting for mass spectrometry
Sample submission form
We urge you to contact facility staff to discuss your project prior to sample preparation.
Identification of proteins from gel bands top
We recommend one-dimensional LC-MS/MS for the identification of proteins from gel bands. Although we can generally make an identfication from any band that can be seen with a protein stain, we recommend a particular stain for best results.
Please refer to the following sample preparation protocols:
Enzymatic digestion of proteins from gel bands
Sample submission form
We urge you to contact facility staff to discuss your project prior to sample preparation.
Custom protocols top
It is also possible to do a variety of other analyses, including isotopic/metabolic analysis.
Although the PMSF has both kinds of sources, protocols using direct ESI and MALDI sources can only be run by special arrangement.
Please contact facility staff to discuss custom protocols.
|
