Sample preparation (mass spectrometry)
Encyclopedia
The sample preparation for mass spectrometry is used for the optimisation of a sample for the analysis in a mass spectrometer (MS).
These have depending on their ion source
Ion source
An ion source is an electro-magnetic device that is used to create charged particles. These are used primarily to form ions for mass spectrometers, optical emission spectrometers, particle accelerators, ion implanters and ion engines.- Electron ionization :...

 different requirements for volume, concentration
Concentration
In chemistry, concentration is defined as the abundance of a constituent divided by the total volume of a mixture. Four types can be distinguished: mass concentration, molar concentration, number concentration, and volume concentration...

, and composition of the analyte
Analyte
An analyte, or component , is a substance or chemical constituent that is of interest in an analytical procedure. Grammatically, it is important to note that experiments always seek to measure properties of analytes—and that analytes themselves can never be measured. For instance, one cannot...

 solution. Furthermore, in protein mass spectrometry
Protein mass spectrometry
Protein mass spectrometry refers to the application of mass spectrometry to the study of proteins. Mass spectrometry is an important emerging method for the characterization of proteins. The two primary methods for ionization of whole proteins are electrospray ionization and matrix-assisted laser...

 usually the protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

 of interest is cleaved into peptides before analysis, either by in-gel digestion
In-gel digestion
The in-gel digestion is part of the sample preparation for the mass spectrometric identification of proteins in course of proteomic analysis. The method was introduced 1992 by Rosenfeld...

 or by proteolysis
Proteolysis
Proteolysis is the directed degradation of proteins by cellular enzymes called proteases or by intramolecular digestion.-Purposes:Proteolysis is used by the cell for several purposes...

 in solution.

Matrix-assisted laser desorption/ionization

For matrix-assisted laser desorption/ionization
Matrix-assisted laser desorption/ionization
Matrix-assisted laser desorption/ionization is a soft ionization technique used in mass spectrometry, allowing the analysis of biomolecules and large organic molecules , which tend to be fragile and fragment when ionized by more conventional ionization methods...

 (MALDI) mass spectrometry the sample is usually mixed with a matrix
Matrix (chemical analysis)
In chemical analysis, matrix refers to the components of a sample other than the analyte. The matrix can have a considerable effect on the way the analysis is conducted and the quality of the results obtained; such effects are called matrix effects. For example, the ionic strength of the solution...

 solution and spotted on the target plate. The matrix crystallises
Crystallization
Crystallization is the process of formation of solid crystals precipitating from a solution, melt or more rarely deposited directly from a gas. Crystallization is also a chemical solid–liquid separation technique, in which mass transfer of a solute from the liquid solution to a pure solid...

 together with the sample and the analyte molecule
Molecule
A molecule is an electrically neutral group of at least two atoms held together by covalent chemical bonds. Molecules are distinguished from ions by their electrical charge...

s are transferred to the gas phase by the pulsed laser
Laser
A laser is a device that emits light through a process of optical amplification based on the stimulated emission of photons. The term "laser" originated as an acronym for Light Amplification by Stimulated Emission of Radiation...

 irradiation.

The concentration of salt in the sample is thereby not so critical as it is for electrospray ionization
Electrospray ionization
Electrospray ionization is a technique used in mass spectrometry to produce ions. It is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when ionized...

. However, interfering signals are observed due to side reactions of the matrix with alkali metal
Alkali metal
The alkali metals are a series of chemical elements in the periodic table. In the modern IUPAC nomenclature, the alkali metals comprise the group 1 elements, along with hydrogen. The alkali metals are lithium , sodium , potassium , rubidium , caesium , and francium...

 ions which can impair the analysis of the spectra. Nevertheless, a separate desalting step is usually not necessary as in previous steps of sample preparation e.g. in-gel digestion
In-gel digestion
The in-gel digestion is part of the sample preparation for the mass spectrometric identification of proteins in course of proteomic analysis. The method was introduced 1992 by Rosenfeld...

 of protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

s, the selection of appropriate buffer
Buffer solution
A buffer solution is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. It has the property that the pH of the solution changes very little when a small amount of strong acid or base is added to it. Buffer solutions are used as a...

 salts prevents the occurrence of this problem. Furthermore the crystallised matrix/analyte mixture allows removing of salts by washing on the target and the addition of ammonium phosphate
Ammonium phosphate
Ammonium phosphate is the salt of ammonia and phosphoric acid. It has the formula 3PO4 and consists of ammonium cations and phosphate anion. It is obtained as a crystalline powder upon mixing concentrated solutions of ammonia and phosphoric acid, or on the addition of excess of ammonia to the...

 before crystallisation can improve the signal quality.

MALDI matrices used for identifying peptides include CHCA and DHB. These solutions are typically saturated solutions in a 50% Acetonitrile/0.1% TFA; TFA being the protonating agent. Typically the sample solution and matrix solution is mixed in a 1:1 ratio and then a half a microliter to a microliter is added to the MALDI plate. Proteins use SA (sinapinic acid) as their matrix; which is prepared in the same way as the peptide matricies.

Electrospray ionization

Electrospray ionization
Electrospray ionization
Electrospray ionization is a technique used in mass spectrometry to produce ions. It is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when ionized...

 because of its principle of ionization is more demanding in the characteristics of the sample. In off-line measurements (in contrast to on-line measurements using LC-MS), the analyte solution is applied by a spray capillary to the mass spectrometer. This capillary allows for the application of volumes in the nanoliter range; however, the detection limit of the instrument requires in protein analysis a relatively high concentration of the analyte. Therefore, usually a step to increase the concentration of the sample is needed.

The evaporation of the solvent in the spray during the ionization process accounts for the demand of a salt-free analyte solution. Otherwise, with the continuous reduction of the volume of the droplets in the spray the salts would start to aggregate which inhibits the formation of useful analyte ions for the measurement.

A major application for ESI is the field of protein mass spectrometry
Protein mass spectrometry
Protein mass spectrometry refers to the application of mass spectrometry to the study of proteins. Mass spectrometry is an important emerging method for the characterization of proteins. The two primary methods for ionization of whole proteins are electrospray ionization and matrix-assisted laser...

. Here, the MS is used for the identification and sizing of proteins. The identification of a protein sample can be done in an ESI-MS by de novo sequencing (using tandem mass spectrometry
Tandem mass spectrometry
Tandem mass spectrometry, also known as MS/MS or MS2, involves multiple steps of mass spectrometry selection, with some form of fragmentation occurring in between the stages.-Tandem MS instruments:...

) or peptide mass fingerprinting
Peptide mass fingerprinting
Peptide mass fingerprinting is an analytical technique for protein identification that was developed in 1993 by several groups independently. In this method, the unknown protein of interest is first cleaved into smaller peptides, whose absolute masses can be accurately measured with a mass...

. Both methods require the previous digestion of proteins to peptides, mostly accomplished enzymatically using protease
Protease
A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain forming the protein....

s. As well for the digestion in solution as for the in-gel digestion
In-gel digestion
The in-gel digestion is part of the sample preparation for the mass spectrometric identification of proteins in course of proteomic analysis. The method was introduced 1992 by Rosenfeld...

 buffered solutions are needed, whose content in salts is to high and in analyte is to low for a successful ESI-MS measurement. Therefore, a combined desalting and concentration step is performed. Usually a reversed phase liquid chromatography
High-performance liquid chromatography
High-performance liquid chromatography , HPLC, is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.HPLC typically utilizes different types of stationary...

 is used, in which the peptides stay bound to the chromatography
Chromatography
Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures....

 matrix whereas the salts are removed by washing. The peptides can be eluted from the matrix by the use of a small volume of a solution containing a large portion of organic
Organic compound
An organic compound is any member of a large class of gaseous, liquid, or solid chemical compounds whose molecules contain carbon. For historical reasons discussed below, a few types of carbon-containing compounds such as carbides, carbonates, simple oxides of carbon, and cyanides, as well as the...

 solvent, which results in the reduction of the final volume of the analyte. In LC-MS the desalting/concentration is realised with a pre-column, in off-line measurements reversed phase micro columns are used, which can be used directly with microliter pipettes. Here, the peptides are eluted with the spray solution containing an appropriate portion of organic solvent. The resulting solution (usually a few microliters)is enriched with the analyte and, after transfer to the spray capillary, can be directly used in the MS.
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