ThermoFisher LTQ mass spectrometer
The ThermoFisher LTQ (pictured above) is a segmented linear ion trap mass spectrometer equipped with an electrospray ionization source interchangeable with a nanospray source. The Proteomics Laboratory has the LTQ interfaced to a ThermoFisher Surveyor HPLC system (lower left) and a SparkHolland Micro Autosampler (upper left). The Surveyor is a quaternary, low-pressure mixing pump with vacuum degassing and delivers flow rates from 250 to 2000 µl/min. In the configuration shown above the flow rate is split to produce an exit flow from the tip of a C18 capillary column into the LTQ of 150-250 nl/min (adjustable to suit the application). The low flow rate allows for low concentrations of analyte to be delivered over sustained periods of time to maximize signal and hence detection. The Micro Autosampler can deliver reproducibly injections of as low as 1 µl of sample into the capillary column. Protein samples in solution are introduced into the mass spectrometer in a fine spray from the tip of a capillary reversed phase (C18) column and the instrument can be set up to collect full scan MS and MSn (n=2-10) data. Advanced scanning modes include data-dependent scans, zoom scans, and ultra zoom scans. Ion mapping experiments can be used to generate full scan, neutral loss, and parent ion maps. Both positive and negative ions can be detected. The sytem (LTQ, Surveyor, and Microautosampler) are controlled by the Xcalibur software which also collects data produced during each run.
Protein identification by Mass Spectrometry
In the LTQ system protein identification is most efficiently achieved by the procedure known as protein mass fingerprinting (PMF). In PMF proteins in solution are denatured, reduced, alkylated (to prevent reoxidizing of disulphide bridges) and digested with a protease (most commonly trypsin). Proteins in gel bands or spots are reduced, alkylated and digested in the gel piece and tryptic peptides encouraged to diffuse out of the gel. After desalting the peptides are sprayed into the LTQ as they elute from a C18 column.
The many thousands of spectra that are produced during a typical run are analyzed by the SEQUEST algorithm and compared in silico with theoretical spectra created from a protein database and the quality of fit rated based on criteria of which some are user determined.
The figure below shows a total ion chromatogram such as is produce by the Xcalibur data collection software and a small selection of data matches produced by the SEQUEST algorithm which is part of the Bioworks suite of protein identification software for which the Proteomics Laboratory has a full license and a dedicated analysis workstation.
The left hand side column shows scan number and the charge of the parent peptide from which the particular spectrum was derived. The center panel shows the total ion chromatogram for all the peptides in the run. The lower panel shows a small selection of proteins to which the sequence of one or more peptides in the mixture corresponds.
BeckmanCoulter ProteomeLab PF2D
The ProteomeLab PF2D is an integrated chromatography system composed of a high performance single-pump chromatofocusing unit (1st of the white units from the left hand side in the figure above) connected to a SparkHolland Micro fraction collector/autosampler (shorter unit next in line to the right) which is interfaced with a dual pump high performance liquid chromatography system which delivers fractions to a large-bed fraction collector capable of holding up to 8 collection plates. The PF2D system is designed for high-resolution analysis of complex protein mixtures. The PF2D automatically separates proteins and collects data from two dimensions by initially separating the proteins on a chromatofocusing column then applying the fractions from the first column to a highly resolving non-porous reversed phase chromatography column. Fractionation on the first column is monitored by an in-line pH meter so that fractions from two related samples (such as treated and untreated cultured cells) can be compared against the pH range from which they were derived prior to reversed phase separation. The software produces detailed reversed phase chromatograms which can be overlaid to distinguish differences in protein expression between the two proteomes as shown in the figure below.
In the figure above chromatograms from two comparable proteomes (the total dissolved proteome from untreated cultured cells on the left hand side and a similar mass of total dissolved proteome from a population of the same cell type treated with a drug is shown on the right hand side) have been overlaid to emphasize their differences. The three center panels show a digitization of the chromatograms to which they are closest while the middle panel displays a difference map showing where peak areas differ in the two populations. Above the panels (not shown in the figure) is a table showing the characteristics of all displayed peaks and a difference table and a grid position to locate the well in which the sample is to be located.
Akta Explorer 100
The Akta Explorer 100 is a four-pump, high speed liquid chromatography system supplied by GE Healthcare that is driven by Unicorn software both of which are used in many pharmaceutical labs. This very versatile system allows for rapid screening of media and methods to develop and optimize purification strategies for proteins from sources of varying complexity.
Electrophoresis equipment for pouring and running gels of various sizes. BioRad Protean IEF cell for isoelectric focusing of 2D gels. Phastgel system for running highly resolving mini checking gels which are also useful for publication figures.