Chemistry: analytical and immunological testing – Nuclear magnetic resonance – electron spin resonance or other...
Reexamination Certificate
2001-08-13
2004-05-11
Gakh, Yelena G. (Department: 1743)
Chemistry: analytical and immunological testing
Nuclear magnetic resonance, electron spin resonance or other...
C326S131000
Reexamination Certificate
active
06734025
ABSTRACT:
This invention relates to processes of measuring molecular mass by mass spectrometry, in particular to methods of precise calibration of mass spectrometers, and to kits and systems for use in calibrating mass spectrometers.
In recent years the required precision for measurements obtained by mass spectrometry has increased greatly. It is now routine that mass spectrometric data should have error not more than 1 to 5 ppm. Thus it has become increasingly important to obtain accurate calibration of mass spectrometers.
It is known to calibrate mass spectrometers by including calibration compounds of known molecular mass in the sample to be analysed. An ideal set of calibration compounds would include at least two compounds of different molecular mass, one above, one below and both close to the expected molecular mass of the material whose exact mass is to be measured. It is important that compounds of known mass have mass close to the mass to be measured, since the calibration curve is not linear.
Various types of compound are known for use as calibration compounds. These include certain sugar derivatives which can be provided with a range of molecular masses. However, they are difficult to synthesise and tend to break down during the mass spectrometry process into by-products which result in significant background noise in the spectrum. They tend also not to be particularly good flyers in the mass spectrometer.
Alternative systems are based on peptides. However these are again expensive to make. Additionally they tend to have different signal intensities and can be difficult to find in the final spectrum.
It would be desirable to provide methods and systems for calibration of mass spectrometers which include calibration compounds of accurate and predetermined molecular mass, which fly well in the mass spectrometer and which do not give rise to breakdown products which produce background noise in the spectrum. It would also be desirable to provide calibration compounds which are straightforward and inexpensive to make.
According to the invention we provide a method of measuring the molecular mass of a compound Y of unknown molecular mass by mass spectrometry, comprising
providing a sample of compound Y,
providing samples of at least two different compounds each of formula (I), R-X in which R is a trityl group and X is cleavable to form a charged species for mass spectrometry,
and recording the molecular mass of compound Y and the at least two compounds of formula (I) in a mass spectrometer.
Thus in the invention we use the trityl compounds of formula (I) as calibration compounds in mass spectrometry. We find that the trityl groups are particularly suitable for use as calibration compounds. The trityl groups are readily cleaved from the compound of formula (I) by illumination with a laser in the mass spectrometer. Further, the resulting positively charged carbonium ion is very stable and thus sensitivity of detection is high. They also fly well in the mass spectrometer. Furthermore, the unique structure of the trityl group presents scope for a wide range of predetermined molecular masses which can be selected an controlled as appropriate depending upon the estimated molecular mass of the compound to be measured. Generally in the process the compound Y is not also of the formula R-X.
The calibration compounds are of formula R-X in which R is a trityl group. Generally R is R
1
R
2
R
3
C— wherein R
1
, R
2
and R
3
are the same or different and each is a monocyclic or fused ring aromatic group that is substituted or unsubstituted. Preferably each of R
1
, R
2
and R
3
is aryl, preferably phenyl.
Preferably at least one of R
1
, R
2
and R
3
carries a substituent selected from C
1
-C
20
alkoxy or hydrocarbyl, substituted or unsubstituted. Substituents may be present at any point in the aromatic ring, but parka substituents are convenient and preferred.
When the alkoxy or hydrocarbyl is substituted the substituent is preferably selected from the group consisting of carboxylic acid, sulphonic acid, nitro, cyano, hydroxyl, thiol, primary, secondary or tertiary amino, primary or secondary amido, anhydride, carbonyl halide and active ester. In these substituents, hydrogen atoms may be partly or wholly replaced by deuterium or halogen, e.g., fluorine. This improves the range of molecular weights available. For instance, alteration of the molecular mass may be obtained by the use of non-radioactive isotopic substituents, e.g., small alkyl groups containing 1, 2 or 3 deuterium atoms.
It is preferred that the trityl group R is substituted with amide substituents. Substituted trityl groups of this type are particularly easy to produce, with a wide range of molecular masses. Thus preferably the trityl group R includes two or four or more amide substituents. More preferably R
1
, R
2
and R
3
together carry at least two amide groups and/or at least two reactive groups for coupling, preferably N-hydroxy hydroxysuccinimide ester groups.
The group X is cleavable to form a charged species for mass spectrometry. Usually it is photocleavable, e.g., by laser illumination. X may be any suitable leaving group, for instance halide or tosylate.
In the method at least two different compounds of formula (I), having different molecular mass, are used. Preferably the number of compounds of formula (I) is larger, for instance at least 5, preferably at least 10 and even at least 20 may be desirable. The compounds of formula (I) are generally selected to have a range of molecular masses around the estimated molecular weight of compound Y. Desirably the molecular masses of the compounds of formula (I) are close to that of compound Y, for more accurate calibration. Thus in a preferred method according to the invention the molecular mass of unknown compound Y is estimated as M
y
and at least one compound of formula (I) having known molecular mass M
1
below M
y
is provided, as is at least one different compound of formula (I) having molecular mass M
2
above M
y
. Preferably each of M
1
and M
2
lies in a molecular mass range not more than ±50% of M
y
. That is M
1
is not less than 50% of M
y
and M
2
is not greater than 150% of M
y
. Preferably the range is within ±25%, more preferably ±10%.
It is possible to provide prepared mixtures of at least two compounds of formula (I) which can be combined with a sample Y of unknown molecular mass and used as calibration compounds in mass spectrometry. Such mixtures preferably comprise at least 5, more preferably at least 10 and in some cases at least 20 different compounds of formula (I).
Thus in a second aspect of the invention we provide a method of measuring the molecular mass of a compound Y of unknown molecular mass comprising estimating the expected molecular mass of compound Y, selecting at least one calibration compound of formula (I) R-X having molecular mass close to the expected molecular mass of the compound Y, in which R is a trityl group and X is cleavable to form a charged species for mass spectrometry, and subjecting both compounds to mass spectrometry simultaneously.
In this aspect the compound of formula (I) may have any of the preferred features indicated for the first aspect of the invention above, as appropriate.
According to a third aspect of the invention we provide mixtures suitable for use in mass spectrometry as calibration compounds. According to this aspect we provide a set of calibration compounds for mass spectrometry comprising at least two separately packaged mixtures (a) and (b), wherein
mixture (a) comprises at least two different compounds each of formula (I) R-X and having different molecular masses
mixture (b) comprises at least two further compounds of formula (I) R-X having different molecular masses and wherein R is a trityl group and X is cleavable to form a charged species for mass spectrometry.
Thus the set comprises at least two different sets of calibration compounds. Preferably each set has molecular masses extending across a different range. The ranges may overlap. Thus preferably the lowest molecul
Gakh Yelena G.
ISIS Innovation, Ltd.
Saliwanchik Lloyd & Saliwanchik
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