Proteome Sciences. AGM Statement.

In continuation of 23855 / 23865 / 23868.

Maxwellsdemon 16 Feb'05 - 08:48 - 7314 of 23902 (premium)


New Sensitizer patent (published 10 February 2005) :


(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(11) WO 2005/012914
(13) A2


(21) PCT/GB2004/003139

(22) 22 July 2004 (22.07.2004)

(25) English
(26) English

(30) 0317123.8 22 July 2003
(22.07.2003) GB
(43) 10 February 2005 (10.02.2005)
(51)7 G01N 33/68, 33/533, C07K 1/13
(54) CHARACTERISING POLYPEPTIDES
(71) XZILLION GMBH & CO. KG [DE/DE]; Industriepark Hochst, Building G865, D-65926 Frankfurt am Main (DE). PROTEOME SCIENCES PLC [GB/GB]; Coveham House, Downside Bridge Road, Cobham, Surrey KT11 3EP (GB).
(72)
(75) SCHAFER, Jurgen [DE/DE]; Am Langen Weg 2, D-36341 Lauterbach (DE). HAMON, Christian [FR/DE]; Luthmerstrasse 49, D-65934 Frankfurt am Main (DE). SCHWARZ, Josef [DE/DE]; Voelklinger Weg 7, D-60529 Frankfurt am Main (DE). PEARCE, Christopher [GB/GB]; Coveham House, Downside Bridge Road, Cobham, Surrey KT11 3EP (GB).
(74) HILL, Christopher, Michael et al.; Page White & Farrer, 54 Doughty Street, London WC1N 2LS (GB).
(81) AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN, CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NA, NI, NO, NZ, OM, PG, PH, PL, PT, RO, RU, SC, SD, SE, SG, SK, SL, SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, YU, ZA, ZM, ZW
(84) ARIPO patent (BW, GH, GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European patent (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HU, IE, IT, LU, MC, NL, PL, PT, RO, SE, SI, SK, TR), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG, BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG)

For information on time limits for entry into the national phase please click here
Published
-- without international search report and to be republished upon receipt of that report

(57) Provided is a method for characterising an analyte by matrix assisted laser desorption ionisation (MALDI) mass spectrometry, which method comprises: (a) labelling the analyte with a light-absorbing label that absorbs light at a pre-determined frequency, to form a labelled analyte; (b) embedding the labelled analyte in a matrix formed from at least one compound that absorbs light, to form an embedded labelled analyte; (c) desorbing the embedded labelled analyte by exposing it to light having the pre-determined frequency, to form a desorbed analyte; and (d) detecting the desorbed analyte by mass spectrometry to characterise the analyte; wherein the light absorbing label comprises a fluorophore moiety, and wherein prior to detecting by mass spectrometry, the analyte is selected for detection on the basis of its fluorophore moiety.

- 17 Feb'05 - 13:45 - 7479 of 23902 (premium)


Below I have reproduced some extracts from the latest PRM Sensitizer patent. The label "D" is what I would describe as the Sensitizer tag. I can see why PRM are talking about a Sensitizer family, because it looks as if they are proposing to use the Sensitizer tag "D" not only in conjunction with their own tags TMT, PST etc. but also other proprierty techniques such as DIGE.



.........An improved 2-D gel technology, in which two or more samples are covalently labelled with different fluorophores and then separated on the same gel has been developed (Unlu, M.; Morgan, M. E.; Minden, J. S. Electrophoresis 1997, 18, 2071-2077). This technology, referred to as DIGE allows paired samples to be compared quantitatively in the same analysis, which is highly advantageous as it avoids the reproducibility problems that plague comparisons of samples run on different gels. The fluorescently labelled proteins can be detected without additional staining steps, which are typically required to visualise proteins on gels. Avoiding staining is advantageous as many stains interfere with subsequent analysis by mass spectrometry. The DIGE process however, does not enhance the sensitivity of the MALDI TOF analysis typically used for peptide mass fingerprinting.......


Accordingly, the present invention provides a method for characterising an analyte by matrix assisted laser desorption ionisation (MALDI) mass spectrometry, which method comprises: (a) labelling the analyte with a light-absorbing label that absorbs light at a pre-determined frequency, to form a labelled analyte; (b) embedding the labelled analyte in a matrix formed from at least one compound that absorbs light, to form an embedded labelled analyte; (c) desorbing the embedded labelled analyte by exposing it to light having the pre-determined frequency, to form a desorbed analyte; and (d) detecting the desorbed analyte by mass spectrometry to characterise the analyte; wherein the light absorbing label comprises a fluorophore moiety, and wherein prior to detecting by mass spectrometry, the analyte is selected for detection on the basis of its fluorophore moiety. ..........

.......Thus, the present invention provides a method of enhancing MALDI sensitivity using sensitiser mass tags (SMTS) that allows differential quantitation of analytes (e. g. proteins) from different samples in the same experiment. Quantitation may be achieved in a number of ways. These include comparison of the fluorophore fluorescence intensity on a gel or in an HPLC run; comparison of peak height/area in the MALDI spectrum; and inclusion of a mass reporter group onto a label on the analyte and measuring the quantity on the basis of the reporter group identified in a tandem mass spectrometric analysis..........


......In a particularly preferred embodiment, the above method comprises, prior to detecting by mass spectrometry, selecting the analyte for detection on the basis of the identity, and/or quantity of its fluorophore moiety. Thus, for example, when a single sample is to be analysed, the proteins in the sample may be labelled as described above and first separated on a gel (e. g. on the basis of size AND/OR isoelectric point). Normally, it is not desirable to try to identify every protein revealed on the gel, but to be selective. For example, only highly expressed proteins may be of interest. In that case, the quantity of fluorophore may be used to select the proteins to extract from the gel and identify by digestion followed by mass spectrometry. Alternatively, if there are several samples being analysed together, fluorophores may be used to distinguish proteins from different samples and selection may be made on this basis. Of course, the quantity and identity may form the basis of selection if desired.......


......Further provided by the invention is a labelled analyte compound, which compound has either of the following structures: F-D-L-A D-F-L-A wherein F comprises a fluorophore, D comprises a light absorbing label, L comprises a linker and A comprises an analyte. Typically, the fluorophore F is attached to D via a further linker.

If desired, a mass marker M may be situated between D or F and L (F-D-M-L-A, or D-F-M-L-A), especially if the label is itself to be analysed by mass spectrometry.

Also provided is a compound for labelling an analyte, which compound has either of the following structures: F-D-L-R D-F-L-R wherein F comprises a fluorophore, D comprises a light absorbing label, L comprises a linker, and R comprises a reactive functionality for attaching the compound to an analyte. Typically, the fluorophore F is attached to D via a further linker. If desired, a mass marker M may be situated between D or F and L (F-D-M-L-R, or D-F-M-L-R), especially if the label is itself to be analysed by mass spectrometry.

In the case of all of the above types of compound, it is generally preferred that IF M is present, each label has the same M, or at least an M having the same mass, so that the spectrum is not complicated by multiple peaks derived from multiple masses for M...........


.........True liquid matrices are also known such as nitrobenzoyl alcohol. Both types of matrix have some advantages in terms of sample consistency, stability under vacuum and ease of handling however solid matrices still tend to be more sensitive. In the context of the present invention, the improvements in sensitivity may justify the use of liquid matrices. This may have advantages in the automation of sample preparation, as liquid handling robotics are widely available and the use of solutions of matrices, for solid matrix co-crystallisation, which readily clog dispensing devices can be avoided........

See:

DIGE: Link1. Link2

Patent text:
Full patent text

pxpx-17 Feb'05 - 15:07 - 7491 of 23902


maxwellsdemon (or anyone else inclined to answer!!)

please forgive my ignorance of this particular science, but would you mind paraphrasing the significance of the pieces that you highlighted in 7479?

I took the piece in red to be a negative since my understanding of the main advantage of sensitizer was the ability to enhance the sensitivity (by up to 100x)

Or is the point of this patent to " allows differential quantitation of analytes (e. g. proteins) from different samples in the same experiment"

thanks in advance
pxpx

maxwellsdemon 17 Feb'05 - 16:30 - 7502 of 23902 (premium)


pxpx,

No the bit in red is positive with respect to PRM.

PRM had previously filed a patent in which they describe what I call "Sensitizer". For MALDI normally peptides/proteins or analytes are embeded in a matrix. A pulse of light is fired on the matrix and peptides/proteins or analytes are desorbed from the surface. Ideally what you would want to happen is for each desorbed molecule to be ionized and preferably singly ionized but this doesn't happen. Peptides containing arginine end up preferentially ionized vis-a-vis non-arginine containing peptides. Further not all the molecules are ionized. As far as I am aware most are not ionized.

To improve the efficiancy one wants to increase the fraction desorbed molecules which are ionized and to have a uniform fraction of ionization over the group of peptides under test.

To do this PRM/Xzillion ( I am guessing it was actually Andrew Thompson) came up with the idea of tagging on a dye to the peptides which absorbed light of the wavelengths of the laser light. Tagging on this dye greatly improved the fraction of desorbed peptides/molecules which were ionized.

The DIGE (exclusively licenced to Amersham from Carnegie Mellon Univ. Not a PRM technology) system provides a method of labelling peptides with fluorophores. One might label peptides from a diseased patient with one fluorophore and peptides from an undiseased patient with another fluorophore of differening wavelength. You them mix the samples together and separate the mixture using a 2D gel. You can locate the spots on the 2D gel because they will fluoresce. And, because the diseased sample fluoresces at a different wavelength to the undiseased one can make a quantative assesment of the differences in expression between the diseased and undiseased states for each individual spot on the 2D gel.

Having identified the spots that show differences in expression between the diseased and nondiseased samples then one can excise these spots ( and optional digest the sample) for further analysis in a mass spectrometer using MALDI to determine the precise nature of the peptide. Now this is where the PRM invention comes in.

On the simplest level one might just add the Sensitizer tag. This would actually have been done at the earlier stage. So a combined DIGE/sensitizer tag would have been used. This should result in much greater sensitivity (i.e. higher levels of ionization) in the mass spectrometer than would have been the case with the DIGE fluorophores alone.

SO PRM's addition to DIGE should improve DIGE's performance in the mass spectrometer / MALDI phase.

But, the above is just one aspect of the patent application. So it is worth having a read of the whole thing.