Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2001-04-09
2003-09-30
Leary, Louise N. (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S004000, C435S283100, C435S288300, C435S288400, C250S288000
Reexamination Certificate
active
06627410
ABSTRACT:
FIELD OF INVENTION
The invention relates to methods and devices used for digesting small amounts of protein in tiny cut gel pieces and for extracting the peptides resulting from the digestion in preparation for analysis by mass spectrometry.
The invention involves digesting proteins using enzymes within the gel pieces in vessels which have permeable but lyophobic bases in such a manner that they scarcely touch the walls of the vessel, and then rapidly removing the digested proteins from the gel pieces almost completely by gentle centrifuging. It is then advantageous to bond the peptides reversibly to suitable surfaces as quickly as possible. For this purpose, the bases of the vessels may contain structures for bonding the peptides which are suitable for washing and subsequently eluting the peptides. A number of vessels can be combined together to form plates which, for example, can have the size of microtiter plates.
PRIOR ART
Two-dimensional gel electrophoresis is still one of the best and most widely used methods for separating the proteins of a cell aggregate—the so-called “proteome”. When used after enzymatic digestion of proteins to peptides, mass spectrometry is the most sensitive methods for identifying individual proteins and determining their structure. The method itself and the difficulties encountered in using it will be briefly described in the following.
After separation in the gel, the proteins are stained and small samples of the gel containing the protein of interest are cut or punched out around the stain site. The gel samples are placed in a vessel and the stain is removed. Topping up with an enzyme solution (such as trypsin) leads to selective digestion at the cleavage sites determined by the enzyme. When using trypsin, which cleaves the molecule at two specific amino acids, the digestion produces peptides with a broad molar mass distribution around an average of approx. 1000 atomic mass units. The protein is usually clearly characterized by the exact masses of the peptides produced by the digestion. The peptides are able to diffuse inside the gel and slowly migrate out of the gel into the surrounding liquid within a few hours.
The peptides in the fluid are purified and analyzed by a suitable method of analysis using mass spectroscopy. In this method, the molecules are usually ionized by so-called Matrix Assisted Laser Desorption Ionization (MALDI) and the precise masses of the peptides resulting from digestion are measured in a time-of-flight (TOF) mass spectrometer. Other methods of ionization are known and are used, usually with confirmation from other types of mass spectrometer. For the MALDI-TOF analysis, the peptides produced by digestion are introduced on suitable sample carriers into small crystals of matrix substances and bombarded with laser pulses in a mass spectrometer. Their precise masses are determined by the flight time of the ions in the TOF mass spectrometer.
Even if some of the peptides are lost during the processing stage, precisely measured values of the peptides which are still available usually result in clear identification of the protein when a suitable program is used to search the protein sequence databases. It is possible to clarify ambiguous identifications by extensive measurement of the fragments of individual peptides produced by using special methods and elucidating them via their internal structure. This form of identification means that the protein, if known, can be characterized by its name, code, origin and molecular structure.
Details on the measurement of fragmentation and other methods for de-novo sequencing of proteins will not be given here.
Although such a straightforward method for the identification of proteins has been the subject of interest for the past few years, interest is now increasingly being directed toward the differences found between the proteins examined and those in the database. These differences, which relate to the mutative or post-translational changes in the proteins, are and will be the focus of interest in the future. For this, it is not only necessary to be able to measure only a few peptides, which in most cases is sufficient for identification purposes alone, but all possible peptides produced by the digestion. In the methods briefly described above however, many peptides are lost due to their being adsorbed on the walls of the vessel which contains them.
At the same time, it is not only the very concentrated proteins which are present in the gel at concentrations ranging from 10 to 100 picomol which are of interest, but also those at lower concentrations ranging from 10 to 100 femtomol. Now if, for example, 20 femtomol of a peptide is present in 20 microliters of liquid, the individual molecules produced by the digestion process will diffuse freely throughout the liquid and will come into contact with the walls of the vessel many times over within a matter of hours. In a small vessel of around three millmeters diameter, they will come into contact with a wall surface area of around 40 mm
2
. If the wall selectively adsorbs one of the peptides, then that peptide will cover the whole surface with a layer which is at least molecule deep. This monomolecular layer could adsorb approx. 40 picomol of a peptide of 1,000 atomic mass units, i.e. 2,000 times the amount which is available in the solution in our example. Even if the adsorptivity of the vessel wall could be reduced to one thousandth by taking the appropriate measures, the peptide of interest could still be fully adsorbed onto the surface and, if the adsorption bond is irreversible as is frequently the case, there is no way that the peptide could be brought into solution again.
The 20 microliter solution sample used in the example calculated here is currently regarded as large. Converting this sample size to a 1 microliter or 100 nanoliter sample (for the same concentration of test molecule) would increase the effect of coming into contact with the wall dramatically.
In modern biochemistry and molecular biology, samples are typically processed in large numbers simultaneously. A visible exponent of this development is the so-called microtiter plate consisting initially of 96, then 384 and now 1,536 reaction vessels. Recently, a NanoWell™ plate was introduced with 3,456 reaction vessels. Increasing the number still further and providing the tools for processing is only a question of time. Appropriate pipetting and processing robots with storage positions for many microtiter plates, barcode labeling, multi-pipette heads and multi-dispensing systems have been developed for microtiter plates which have been in common use until now.
At the same time, the quantities of sample molecules required for the chemical, enzymatic and analytical processing stage are getting increasingly smaller so that proteins at very low concentration can also be measured. Processing has long since moved from the nanomol range to the picomol, femtomol and even attomol range. The disadvantage of this is that, as the amounts of test solution are progressively reduced, the wall area of the cavities enclosing the liquid progressively increases in relation to their volume and the chemical and physical effects of the cavity walls on the reactions during processing become more critical.
The microtiter plate also forms the ideal basis for processing the proteins of a proteome, for example, in association with an automated gel sampler. Until now, however, microtiter plates have whisked away a large proportion of the peptides produced by the digestion.
OBJECTIVE OF THE INVENTION
The objective of the invention is to find methods and devices to be used for preparing small samples of protein from pieces of gel for analysis using mass spectrometry but which are characterized by high yields for all peptides and particularly low levels of peptide loss for those peptides which are at risk through wall adsorption.
SUMMARY OF THE INVENTION
In detail, the method of the invention uses tiny gel pieces containing protein and relates to the enzymatic digestion of proteins within the gel wh
Meyer Helmut
Nordhoff Eckhard
Schurenberg Martin
Bruker Daltonik GmbH
Leary Louise N.
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