Identification of proteins in protein mixtures.
Identification of
protein modifications
Ordering information:
All services are available to UC Berkeley reserchers on
a recharge basis. Others may purchase services directly. All potential
users are urged to contact facility staff to discuss their project before
subimitting samples.
Sample preparation tips
Mass Spec Facility Recharge Rates-UC Berkeley Community
1D Run (RP LC-MS/MS)...............................................................................................$150/run
2D “MudPIT” Run (cation exchange/RP LC-MS/MS)..................................................$300/run
3D “MudPIT”plus on-column desalting (RP/cation exchange/RP LC-MS/MS).........$400/run
Sample Prep (researchers are urged to do their own preps)
Supply of reagents to researchers
Aliquot of trypsin or other protease....................................... $4
Spec tip for sample desalting................................................ $8
Desalting of a sample by spec tip......................................................$50
Single enzyme digestion.....................................................................$100
Triple enzyme digestion......................................................................$200
Trypsinization and sample recovery from a gel band.....................$200
Mass
Spec Facility Recharge Rates-Non UC Berkeley Community
All
Fees Doubled
Sample submission form
Identification of proteins in protein mixtures.
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.
The desalted, proteolysed mixture is loaded 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 each major ion 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 prepare for this type of analysis, please refer to the following forms and protocols:
Enzymatic digestion of protein samples
Sample desalting for mass spectrometry
We urge you to contact facility staff to discuss your project prior to sample preparation.
Identification of protein modifications
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 multi dimensional “MudPit” chromatography depending on the sample complexity. Often modifications of interest can be found simply by searching LC mass spec data collected under standard conditions.
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 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
We urge you to contact facility staff to discuss your project prior to sample preparation.
Identification of proteins from gel bands
We recommend one-dimensional LC-MS/MS for the identification of proteins from gel bands. Please refer to the following sample preparation protocols:
Enzymatic digestion of proteins from gel bands
We urge you to contact facility staff to discuss your project prior to sample preparation.
Protocols using direct ESI and MALDI sources can be run by special arrangement.
We can also accommodate many custom LC protocols, and limited isotopic analysis is possible.
Please contact facility staff to discuss custom protocols.
Having a protein related research problem? CRL-PMSF staff are expert in protein purification, solubility, and chemical modification. Contact us to discuss your project.
Sample preparation tips
The electrospray ionization method we use requires that the sample be free from salts and from substances such as detergents that suppress ionization. Many affinity tag procedures and kits have detergents in their buffers! If you can’t avoid detergents in your elution buffers, they must be removed prior to enzymatic digestion of the sample. Remember, the ideal sample contains nothing but peptides!
The protocol supplied at the following link is generally useful for removing a variety of small molecule contaminants.