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MudPIT is a non-gel technique for separating and identifying individual components of complex protein and peptide mixtures. The seminal publication on MudPIT showed the potential for applying the technology to proteomic analyses using Saccharomyces cerevisiae and has been cited 1,236 times since its publication.Instead of using traditional ...
Multidimensional Protein Identification Technology (MudPIT) eliminates gel separations. Instead, biochemical fractions containing many proteins are directly proteolyzed and the enormous number of peptides generated, are separated by 2-dimensional liquid chromatography before entering the mass spectrometer.
- Eric C Schirmer, John R Yates, Larry Gerace
- 2003
MudPIT combines both a cation exchange pre-fractionation and RP HPLC separation (1) of tryptic peptides to analyze an entire proteome of a cell or tissue type protein extract.
- Sample Preparation
- Column Preparation and Packing Material
- Sample Application
- HPLC Setup
- Mass Spectrometric Detection
- Data Interpretation
1. Samples for MudPIT analysis should be in an adequate loading buffer system concerning column arrangement without any perturbing salts (see Note 1). If samples are not in adequate loading buffer...2. Loading amount of sample should be around 250 µg for the system described below. Anyway, changing column arrangements or dimensions may require more or less material to be applied. Furthermore,...1. In general, column preparation and dimensions should match to the analytic question. Low sample amounts, for example, do not require excessive lengths of precolumns in this context.2. The column described in this chapter consists of three parts (RP- and SCX-precolumns followed by RP-separation column) packed into a 100 µm ID × 360 µm OD capillary with a nano-emitter tip at on...3. Prepare slurries of 50 mg/400 µL methanol for each packing material, sonicate and vortex thoroughly. Start with preparation of main separation column by placing the RP-slurry within a 1.5 mL rea...4. Prior to initial use, the MudPIT column should be preconditioned and checked by applying a digest of a standard protein or a peptide mixture of known composition. Samples are applied as stated b...1. Prior to use, MudPIT columns should be equilibrated in buffer A for 15 min either by plugging the column to an HPLC system at 250 nL/min or again by placing the column into packing bomb with buf...2. Place up to 50 µL of sample into a reaction tube and load sample within the bomb-packing device to the column by pressurizing. Loading proceeds until column runs dry.1. HPLC system consists of nano-LC, e.g., Ultimate™ (Dionex, Idstein, Germany) or equal with four different solvent channels (for solvents, see Subheading 2 and Note 7). Flow rate for direct coupli...2. The first gradient serves two purposes. First, the column is washed with buffer A for 30 min to remove salts and to condition the SCX-phase. Second, the sample is transferred onto the SCX-precol...3. Subsequently, peptides are stepwise eluted from the SCX-column onto the RP-separation column by applying increasing amounts of buffers C and D. In detail, for each run, the column is equilibrate...For mass spectrometric detection, a LTQ linear ion trap with a nano-ion spray source is used (LTQ XL, ThermoFisher Scientific, Dreieich, Germany). Instrument parameters may vary for different setups; however, in this case, spray voltage of 2 kV is used. All spectra are acquired with normal scan speed. Most commonly, a survey spectrum recorded from ...
In general, peptides of a given protein can elute all over a MudPIT experiment. Therefore, it is mandatory to either search all individual runs and combine the results or to directly search all the combined raw data at once. However, because of the high number of acquired spectra during a MudPIT experiment, usually the former option is chosen, sinc...
- Katharina Lohrig, Dirk Wolters
- 2009
1 de ago. de 2005 · The archetypal approach, termed MudPIT (for Multidimensional Protein Identification Technology) [19], pioneered in the laboratory of John Yates, III, has proven to be a remarkably effective and robust methodology for investigating global changes in protein expression as a function of development and disease 20, 21, 22.
- Thomas Kislinger, Anthony O. Gramolini, David H. MacLennan, Andrew Emili
- 2005
24 de may. de 2009 · Multidimensional Protein Identification Technology (MudPIT) eliminates gel separations. Instead, biochemical fractions containing many proteins are directly proteolyzed and the enormous number of peptides generated, are separated by 2-dimensional liquid chromatography before entering the mass spectrometer.
Mass Spectrometry and Complex Protein Mixture Sequencing (1-3000 proteins) MUDPIT (multidimensional protein identification technology) is the method of choice for complex protein sample analysis in which more elaborate separation techniques are needed.
