Systems and method for discovery and analysis of markers
View Patent ↗The present invention relates to a charged particle beam apparatus which employs a scanning electron microscope for sample inspection and defect review. The present invent provides solution of improving imaging resolution by utilizing a field emission cathode tip with a large tip radius, applying a large accelerating voltage across ground potential between the cathode and anode, positioning the beam limit aperture before condenser lens, utilizing condenser lens excitation current to optimize image resolution, applying a high tube bias to shorten electron travel time, adopting and modifying SORIL objective lens to ameliorate aberration at large field of view and under electric drifting and reduce the urgency of water cooling objective lens while operating material analysis. The present invent provides solution of improving throughput by utilizing fast scanning ability of SORIL and providing a large voltage difference between sample and detectors.
1. A method of identifying candidate biomarkers in the plasma microparticle sub-proteome, comprising: (a) isolating proteins from blood plasma microparticles obtained from at least one blood sample from a case and at least one blood sample from a control; (b) identifying the proteins; (c) applying a statistical test to measure the abundance of each protein identified; and, (d) determining the presence of positive and negative biomarkers by comparing the abundance of each protein identified in at least one case blood sample with the abundance of the same protein in at least one control blood sample.
2. The method of claim 1 , wherein (b) identifying is performed by sequencing the resulting proteins or peptides.
3. The method of claim 2 , wherein (b) identifying is performed by sequencing the resulting peptides.
4. The method of claim 3 , wherein the sequencing is performed by mass spectrometry.
5. The method of claim 4 , wherein the sequencing is performed by a HPLC-MS/MS.
6. The method of claim 1 , wherein (c) applying a statistical test is performed by using a method selected from: ion current, spectral count, and coverage.
7. The method of claim 6 wherein the statistical method used is comparing spectral counts between cases and controls.
8. The method of claim 1 , wherein isolating proteins (a), further comprises:
(e) isolating a plasma microparticle-enriched fraction from a blood sample; (f) purifying plasma microparticles from the enriched fraction; and, (g) isolating proteins from the purified microparticles.
9. The method of claim 8 , further comprising: (l) optionally cleaving the resulting proteins to obtain peptides.
10. The method of claim 9 , wherein (l) cleaving is performed by digesting the delipidated proteins with an appropriate enzyme.
11. The method of claim 10 , wherein the enzyme is trypsin.
12. The method of claim 8 , wherein isolating a plasma microparticle-enriched fraction (e), comprises: (h) separating platelet-poor plasma (PPP) from a blood sample; and, (i) fractionating by size to obtain a plasma microparticle-enriched fraction.
13. The method of claim 12 , wherein (h) separating the plasma microparticle-enriched fraction is performed by centrifugation.
14. The method of claim 12 , wherein (i) purifying by fractionating by size is performed by running the fractionated plasma microparticles through a size exclusion column.
15. The method of claim 12 , wherein purifying plasma microparticles (f), comprises: (j) fractionating the plasma microparticle-enriched fraction to obtain plasma microparticles.
16. The method of claim 15 , wherein (j) fractionating the PPP is performed by centrifugation.
17. The method of claim 16 , wherein the centrifugation is sufficient to form a pellet comprising: plasma microparticles.
18. The method of claim 15 , wherein isolating proteins (g), comprises: (k) delipidating the resulting plasma microparticles to obtain proteins.
19. The method of claim 18 , wherein (k) delipidating is performed by submitting the plasma microparticles to gel electrophoresis.
20. The method of claim 19 , wherein the gel electrophoresis is 1D SDS PAGE.