Liquid Chromatography-Mass Spectrometry (LC-MS)

Thermo Finnigan LCQ Deca Plus ProteomeX Work station.

This is an ion trap type instrument, with MSn capability, interfaced with a 2D-LC. It is primarily designed to analyse complex mixtures of peptides, separating them with a series of salt cuts from a capillary cation exchange column. The individual salt cuts are then further separated on capillary C-18 column.  The MS is equipped with an ESI source fitted with a metal needle kit, a nanoESI source and a combined APCI and APPI source.

Micromass QToF 2
This MS is interfaced to a capillary LC and has both an ESI and nanoESI sources. This is a hybrid instrument with two analysers, a quadrupole and a ToF, separated by a collision cell. It generates MS and MS/MS data with high mass accuracy and mass resolution.

LC-MS allows separation of complex mixtures of non-volatile compounds before introduction to the mass spectrometer. It is used extensively for compounds that have a high molecular weight or are too heat labile to be analyzed by GC-MS. These include peptides, proteins, oligonucleotides, polysaccharides and a variety of primary and secondary metabolites. The most common ionization methods employed for LC-MS are ESI, APCI and APPI in positive and negative-ion modes. The chromatography is, in most cases, reverse phase, and it is important that the eluting solvents and buffers are volatile.

Electrospray Ionisation (available on both LCQ and QToF2)

Electrospray ionization (ESI) transfers ions in solution into the gas phase. The spectrum of higher molecular weight proteins and peptides , for example, typically consist of a distribution of multiply charged analyte ions. ESI can be used for small and large molecular-weight biopolymers (peptides, proteins, carbohydrates, and DNA fragments), and smaller metabolites which are present in solution in the ionised form. ESI generates both positive and negative ions; acidic molecules form negative ions in high pH solution, and basic molecules form positive ion in low pH solution. These pre-formed ions can include adduct ions.

Many LC applications use non-volatile buffers such as phosphate. These may not be used in LC-MS. Separations should be developed using volatile buffers (see note on sources of contamination below):

• Acetic acid
• Formic acid
• Ammonium acetate
• Ammonium formate
• Ammonium hydroxide
• Triethylamine (TEA)
• Trifluoracetic acid (TFA) (not recommended for peptides and proteins)

Unlike MALDI, which is a pulsed technique, ESI is a continuous ionization method that is suitable for using as an interface with HPLC or capillary electrophoresis. ESI should be considered as being complementary to MALDI. The sample must be soluble, stable in solution, polar, and relatively clean (free of nonvolatile buffers, detergents, salts, etc.).

Atmospheric Pressure Chemical Ionisation/ Atmospheric Pressure Photo Ionisation (APPI/APCI)
(available on LCQ only)
The combination of Atmospheric Pressure Chemical Ionisation/ Atmospheric Pressure Photo Ionisation (APPI/APCI) is used for analysis of small molecules that are not readily ionised by ESI (e.g. steroids, basic compounds, and pesticides).

In APCI, the sample solvent is evaporated and passed through a corona discharge. The corona discharge forms reagent ions with solvent molecules and the nitrogen sheath gas. These can then react with the analyte molecules to form ions. The relative gas phase acidity of the reagent ions and the analyte molecules plays an important role in the APCI process. Positive ions are formed by proton adduction (MH+) and negative ions by proton abstraction (MH-).

Primary ion formation:                e- + N2     g     N2+.   +  2e-

Secondary ion formation:    N2+.+ H2O     g     N2   +  H2O+.

                                          H2O+. + H2O     g     H3O+   +  HO.

Proton transfer:                  H3O+   +  M       g     MH+  +   H2O

In the negative ion mode, (M-H)- is typically formed by OH- abstracting a hydrogen.

In APPI, molecular ions are formed when the ionisation potential of the analyte molecule is less than the photon energy of the incident light (10eV) so that a photon displaces an electron.

      M   +   hv     g     M+.   +  e-

In the presence of protic solvents (S) the analyte ion may abstract a hydrogen to form a MH+ ion.

    M+.   +  S     g     MH+  +  (S-H)

In the combined source you can easily change between APCI, APPI and combined APCI/APPI techniques by turning on the corona discharge, the light source or both.

Tandem Mass Spectrometry (MSn)
Tandem MS or MSn is used for structure determination of molecular ions or fragments. In Tandem MS, on the QTof2, the ion of interest is selected with the first analyzer (MS-1, quadrupole)), collided with inert gas atoms (argon) in a collision cell. The fragments generated by the collision are then separated by a second analyzer (MS-2, Tof). The use of multiple analysers is known as tandem MS in space. The Qtof can generate spectra with high mass accuracy but is limited to MS2.

In the ion trap instruments (Polaris Q and LCQ) the experiments are carried out in one analyzer, and the various events are separated in time, not in space. Hence tandem MS in time. Provided there are sufficient ions, MS15 spectra can be obtained but only at unit resolution.

The information generated from these types of experiments can be used to sequence peptides and small DNA/RNA oligomers, to determine structure and connectivity of polysaccharides, to determine the position and structure of fatty acids in complex lipids, and to carry out other structure determinations.

Sources of Contamination in LC-MS

As detectors for HPLC, mass spectrometers represent a substantial increase in sensitivity.  Consequently, analysts need to become aware of the possibility of introducing contaminants into their samples as these can not only mask sections of the chromatogram but can result in a substantial decrease in assay sensitivity.  Contaminants may also affect subsequent samples and result in instrument downtime while they are removed from the system.

Use only solvents, acids, buffers and water that has been prepared specifically for LC-MS.  Detergent residues (Triton, Tween and Decon 90, series of peaks with Dm = 44) and plasticware (dioctyl phthalate, MH+ = 391.28, MNa+ = 413.27) are common sources of contamination.  The former can be eliminated by the use of chromic acid washed glassware, particularly the autosampler vials.   For further information see the following papers and references therein:

Tran and Doucette. Cyclic polyamide oligomers extracted from Nylon66 membrane filter disks as a source of contamination in LC/MS.  Journal of the American Society for Mass Spectrometry (2006) 17, 652-656.

Hao, March Croley, Smith and Rafferty.  Electrospray ionisation tandem mass spectrometric study of salt cluster ions. Part 1 - Investigations of alkali metal chloride and sodium salt cluster ions.  Journal of Mass Spectrometry (2001) 36, 79-96.

Tong, Bell, Tabei and Seigal.  Automated data massaging, interpretation and e-mailing modules for high throughput open access mass spectrometry.  Journal of the American Society for Mass Spectrometry (1999) 10, 1174-1187.

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