MudPIT at the Yates Lab

The MudPIT Page

MudPIT (Multidimensional Protein Identification Technology) is a technique for the separation and identification of complex protein and peptide mixtures. Rather than use traditional 2D gel electrophoresis, MudPIT separates peptides in 2D liquid chromatography. In this way, the separation can be interfaced directly with the ion source of a mass spectrometer.

Technique

MudPIT uses columns consisting of strong cation exchange (SCX) material back-to-back with reversed phase (RP) material inside fused silica capillaries. The chromatography proceeds in cycles, each comprising an increase in salt concentration to "bump" peptides off of the SCX followed by a gradient of increasing hydrophobicity to progressively elute peptides from the RP into the ion source.

The mass spectrometer's data-dependent acquisition isolates peptides as they elute and subjects them to Collision-Induced Dissociation, recording the fragment ions in a tandem mass spectrum. These spectra are matched to database peptide sequences by the SEQUEST algorithm. SEQUEST's peptide identifications are assembled and filtered into protein-level information by the DTASelect algorithm.

Concentrations

A sample set of SCX concentrations used for a twelve-cycle MudPIT follows. The Concentration is the percent of 500 mM ammonium acetate.

Cycle	Duration (min)	Concentration
1	90	0
2	112	4
3	112	8
4	112	10
5	112	12
6	112	15
7	112	20
8	112	30
9	112	40
10	112	50
11	140	75
12	140	100

Nitty Gritty

We use a Sutter Instrument Company P-2000 to produce our tips from fused silica capillaries, usually purchased from Polymicro or Agilent. Our program for the machine reproducibly generates apertures approximately 5 nanometers across from capillaries that have an inner diameter of 100 microns.
Our custom-made "bombs" help us to load packing material and peptides into our columns. An Eppendorff tube is placed inside the bomb, holding the material to be loaded. The bomb is then assembled and the column inserted through the aperture at the top of the bomb into the Eppendorff below. Gas pressure forces the material in the tube into the column.
The material we load into our columns for SCX is made by Partisphere and comes on 5 micron spherical silica beads. For RP we use 5 micron C-18 coated beads from a variety of vendors.
We use Agilent 1100 and ThermoFinnigan Surveyor Quaternary pumps. They are operated at flow rates of 100-200 microliters/min with pre-column splitting of the flow to produce 100-200 nL/min flow rates at the column.
Our ion source for the ThermoFinnigan LCQ is a specialized stage with ample room for our equipment. First, there's a cross linking the column to the pump flow, waste, and electrode. Second, there's an armature with a video camera so we don't have to squint to see the bubbles forming. Thirdly there's a bed to hold the column itself. We can adjust the stage's position relative to the source by the screws to the left of the cross.

References

Eng JK, McCormack AL, and Yates JR 3^rd, An Approach to Correlate Tandem Mass Spectral Data of Peptides with Amino Acid Sequences in a Protein Database. J Am Soc Mass Spectrom 1994, 5: 976-989.
Link AJ, Eng J, Schieltz DM, Carmack E, Mize GJ, Morris DR, Garvik BM, Yates JR 3^rd. Direct analysis of protein complexes using mass spectrometry. Nat Biotechnol. 1999 Jul;17(7):676-82.
Washburn MP, Wolters D, Yates JR 3^rd. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol. 2001 Mar;19(3):242-7.
Lin D, Alpert AJ, and Yates JR 3^rd, Multidimensional Protein Identification Technology as an Effective Tool for Proteomics. American Genomic/Proteomic Technology, 2001 1(1): 38-46. Review.
Tabb DL, McDonald, WH, Yates JR 3^rd, DTASelect and Contrast: Tools for Assembling and Comparing Protein Identifications from Shotgun Proteomics. J. Proteome Res. 2002 1:21-26.