Orbitrap

4 3.1. Reversed-Phase Liquid Chromatography With complex mixtures so prevalent in most application areas, some sort of sample simplification is required before its introduction into the mass analyzer. Liquid chromatography (LC) is one of the methods that became popular because of its ability to seamlessly couple with electrospray and nanoelectrospray ionization techniques thus ensuring a high degree of au tomation and throughput. The employment of reversed-phase (RP) LC coupled to Orbitrap instruments is truly ubiquitous. In proteomics, the majority of routine protein identifications are performed with this technique often following a digestion of gel separated intact proteins or fractionation of complex peptide mixtures after sample digestion using isoelectric focusing or another form of chromatographic separation, such as Strong Cation Exchanger (SCX). It is of note that a comparison of two sample preparation methods, a one- dimensional sodium dodecyl sulfate gel electrophoresis of intact proteins versusisoelectric focusing of enzymatic digest of the sample, showed that both methods delivered a comparable result with no apparent bias for any functional protein class. 32 While C18 columns are used for peptide analyses, C8 or C4 media are usually preferred for larger peptides and proteins. 33 In the area of small molecule analysis, reversed-phase LC is similarly widespread. A detailed study comparing various reversed-phase separation media has been carried out to develop efficient and robust methods for LC/MS-based metabolomic profiling studies. 34 3.2. Ultra-High Pressure Liquid Chromatography The use of ultra-high pressure LC (U-HPLC) in combination with Orbitrap detection, both for peptide and small molecule analyses, is of a particular interest. Fast acquisition rates are required so as to provide sufficient data points across narrow chromatographic peaks. 35 Publications integrating the Orbitrap mass analyzer to U-HPLC cover areas of plasma phospholipids, drug metabolism, pesticides and veterinary dru g residues, and hormones. 24, 36-39 A transient acquisition time of 0.4 s provides a mass resolution of 30,000 at m/z400 (and 25 data points across a peak of width at baseline of 10 s). 38 Acquisition of MS/MS spectra can be conveniently performed either in the parallel-scanning linear ion trap or in the Orbitrap mass analyzer at resolution of 7,500 with a scan time of 0.1 s (i.e., a full scan at resolutio n 30,000 followed by an MS/MS scan at resolution 7,500, resulting in 20 data points collected across a peak). The positive outcome of the above theoretical estimate was verified for metabolic profiling of serum samples. The results confirmed that peak widths of 5-10 s allow the collection of an adequate number of data points across the chromatographic peak while maintaining good sensitivity and mass accu racy. Applied to the analysis of serum, interfacing with U-HPLC delivered linear response to the concentration of metabolites over 3.5 orders of magnitude with limits of detection less than 1μmol/L. 35 3.3. Multidimensional Liquid Chromatography Combinations of several chromatographic media form the basis of multidimensional LC separation strategies used for complex peptide mixture analyses. 40 The separations can be performed either in-line or off-line of the mass spectrometer and frequently involve the use of SCX in combination with RP chromatography. 41 This strategy can also employ U-HPLC to improve peptide identification in proteomic samples. 42 Combining a reverse phase separation with a special chromato- graphic enrichment step(s), such as hydrophilic interaction chromatography (HILIC) is being mentio ned in the context of phosphopeptide analysis and metabolomics (see Sections 4.4 and 7 herein). 3.4. Gas Phase Separation The ultimate goal would be to eliminate separation steps altogether. Gas phase fractionation holds the promise for being a greatly simplified, fully automated approach. Its effectiveness was demonst