Liquid chromatography–mass spectrometry
Thermo Fisher Scientific
|Related||Gas chromatography–mass spectrometry|
Liquid chromatography–mass spectrometry (LC-MS, or alternatively HPLC-MS) is a chemistry technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry. LC-MS is a powerful technique used for many applications which has very high sensitivity and selectivity. Generally its application is oriented towards the general detection and potential identification of chemicals in the presence of other chemicals (in a complex mixture). Preparative LC-MS system can be used for fast and mass directed purification of natural-products extracts and new molecular entities important to food, pharmaceutical, agrochemical and other industries.
For most protein work, the proteins are first digested into small fragments (say , 5-10 amino acids), separated by HPLC (high performance liquid chromatography), and then run individually through the mass spec. Protein sequencing and older protein identification methods also start with proteolytic digestion Endopeptidases that digest at known sites are used, such as trypsin (cleaves after Lys or Arg) and chymotrypsin (cleaves after Phe, Trp, or Tyr). Ionizing the peptide needs to be done rather gently. One common technique is MALDI (matrix-assisted laser desorption/ionization). The proteins are mixed with the matrix molecules, which efficiently absorb the UV laser energy and encourage ionization of the proteins. When irradiated with the laser, they vaporize along with the protein, but their small size makes them easy to detect and ignore. Time-of-flight mass spectrometry is generally used (so the whole thing is MALDI-TOF). The molecular ions are accelerated in an electric field, and the time it takes them to cross a chamber of known length is proportional to their mass (actually, mass to charge ratio [m/z]). This technique works well for the wide range of sizes seen with peptides. Sample comparisons can be done by labeling one sample with a heavy, stable isotope such as 13C or 15N. The samples are mixed before 2D electrophoresis and they co-migrate on the gel. However, mass spectrometry can easily resolve them.
When standard bore (4.6 mm) columns are used the flow is often split ~10:1. This can be beneficial by allowing the use of other techniques in tandem such as MS and UV. However splitting the flow to UV will decrease the sensitivity of spectrophotometric detectors. The mass spectrometry on the other hand will give improved sensitivity at flow rates of 200 μL/min or less.
Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of charged particles. It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and other chemical compounds. MS works by ionizing chemical compounds to generate charged molecules or molecule fragments and measuring their mass-to-charge ratios. In a typical MS procedure:
A sample is loaded onto the MS instrument and undergoes vaporization.
The components of the sample are ionized by one of a variety of methods (e.g., by impacting them with an electron beam), which results in the formation of charged particles (ions).
The ions are separated according to their mass-to-charge ratio in an analyzer by electromagnetic fields
The ions are detected, usually by a quantitative method.
The ion signal is processed into mass spectra.
Additionally, MS instruments consist of three modules:
An ion source, which can convert gas phase sample molecules into ions (or, in the case of electrospray ionization, move ions that exist in solution into the gas phase)
A mass analyzer, which sorts the ions by their masses by applying electromagnetic fields
A detector, which measures the value of an indicator quantity and thus provides data for calculating the abundances of each ion present
The technique has both qualitative and quantitative uses. These include identifying unknown compounds, determining the isotopic composition of elements in a molecule, and determining the structure of a compound by observing its fragmentation. Other uses include quantifying the amount of a compound in a sample or studying the fundamentals of gas phase ion chemistry (the chemistry of ions and neutrals in a vacuum). MS is now in very common use in analytical laboratories that study physical, chemical, or biological properties of a great variety of compounds.
Understandably the interface between a liquid phase technique which continuously flows liquid, and a gas phase technique carried out in a vacuum was difficult for a long time. The advent of electrospray ionization changed this. The interface is most often an electrospray ion source or variant such as a nanospray source; however atmospheric pressure chemical ionization interface is also used. Various deposition and drying techniques have also been used such as using moving belts; however the most common of these is off-line MALDI deposition. A new approach still under development called Direct-EI LC-MS interface, couples a nano HPLC system and an electron ionization equipped mass spectrometer.
LC-MS is very commonly used in pharmacokinetic studies of pharmaceuticals and is thus the most frequently used technique in the field of bioanalysis. These studies give information about how quickly a drug will be cleared from the hepatic blood flow, and organs of the body. MS is used for this due to high sensitivity and exceptional specificity compared to UV (as long as the analyte can be suitably ionised), and short analysis time.
The major advantage MS has is the use of tandem MS-MS. The detector may be programmed to select certain ions to fragment. The process is essentially a selection technique, but is in fact more complex. The measured quantity is the sum of molecule fragments chosen by the operator. As long as there are no interferences or ion suppression, the LC separation can be quite quick. It is common now to have analysis times of 1 minute or less by MS-MS detection, compared to over 10 mins with UV detection.
LC-MS is also used in proteomics where again components of a complex mixture must be detected and identified in some manner. The bottom-up proteomics LC-MS approach to proteomics generally involves protease digestion and denaturation (usually trypsin as a protease, urea to denature tertiary structure and iodoacetamide to cap cysteine residues) followed by LC-MS with peptide mass fingerprinting or LC-MS/MS (tandem MS) to derive sequence of individual peptides. LC-MS/MS is most commonly used for proteomic analysis of complex samples where peptide masses may overlap even with a high-resolution mass spectrometer. Samples of complex biological fluids like human serum may be run in a modern LC-MS/MS system and result in over 1000 proteins being identified, provided that the sample was first separated on an SDS-PAGE gel or HPLC-SCX.
LC-MS is frequently used in drug development at many different stages including peptide mapping, glycoprotein mapping, natural products dereplication, bioaffinity screening, in vivo drug screening, metabolic stability screening, metabolite identification, impurity identification, quantitative bioanalysis, and quality control.
- Gas chromatography–mass spectrometry
- Capillary electrophoresis–mass spectrometry
- Ion-mobility spectrometry–mass spectrometry
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