Mass spectrometry imaging of glycans from tissue sections and improved analyte detection methods

Abstract

The presently disclosed subject matter provides methods using mass spectrometry for direct profiling of N-linked glycans from a biological sample. In addition, the embodiments of the present invention also disclose novel methods, known as targeted analyte detection (TAD), for improving the detection limit of MALDI-MS. These methods take advantage of the carrier effect of the added standard analytes, which occurs due to the generic sigmoidal shape of the calibration curve. The functionality of TAD depends on the relative enhancement of sensitivity over the increase of the standard deviation at the analysis of target analytes with spiking in exogenous concentration. At certain ranges of exogenous concentration, the increment in the sensitivity overcomes the standard deviation, resulting in an improved LOD. Theoretically, exogenous concentrations approximately at 1 LODorig would generate the optimum LOD improvement. TAD is a cost-effective LOD improvement method, which is not limited to a certain group of analytes, or detection methods or instruments. It can be applied to enhance the detection of any analyte with different detection methods, provided that the analyte of interest can be extracted or is available in synthetic form.

1 . A method for direct profiling of N-linked glycans in a biological sample, the method comprising:
 (a) obtaining a biological sample comprising at least one glycoprotein;   (b) denaturing the at least one glycoprotein in the biological sample;   (c) releasing at least one glycan from the at least one glycoprotein;   (d) coating the biological sample with a matrix;   (e) analyzing the at least one glycan using mass spectrometry; and   
       wherein spatial distribution of the at least one glycan is maintained. 
     
     
         2 . The method of  claim 1 , wherein the biological sample comprises a paraffin-embedded tissue. 
     
     
         3 . The method of  claim 1 , wherein the biological sample comprises a formalin-fixed tissue. 
     
     
         4 . The method of  claim 2 , wherein the biological sample is rehydrated. 
     
     
         5 . The method of  claim 1 , wherein the biological sample is deposited on a solid support. 
     
     
         6 . The method of  claim 5 , wherein the solid support allows sectioning so that spatial distribution is maintained. 
     
     
         7 . The method of  claim 1 , wherein denaturing the at least one glycoprotein comprises heating the biological sample and/or incubating the biological sample with a proteolytic enzyme for a sufficient period of time. 
     
     
         8 . The method of  claim 1 , wherein releasing the at least one glycan occurs by using an enzyme selected from the group consisting of Endoglycosidase H (Endo H), Endoglycosidase F (EndoF), and N-Glycanase F (PNGaseF). 
     
     
         9 . The method of  claim 8 , wherein releasing the at least one glycan occurs by using the enzyme PNGaseF. 
     
     
         10 . The method of  claim 1 , wherein releasing the at least one glycan occurs by using a microarray printer. 
     
     
         11 . The method of  claim 1 , wherein the matrix comprises 2,5-dihydroxybenzoic acid (DHB). 
     
     
         12 . The method of  claim 1 , wherein the mass spectrometry is MALDI-mass spectrometry. 
     
     
         13 . The method of  claim 12 , wherein the MALDI-mass spectrometry comprises MALDI time-of-flight (TOF) mass spectrometry. 
     
     
         14 . The method of  claim 12 , wherein the MALDI-mass spectrometry comprises MALDI-quadrupole ion trap (QIT)-TOF mass spectrometry. 
     
     
         15 . The method of  claim 1 , the method further comprising:
 b1) adding to the sample a known concentration of at least one glycoprotein;   e1) calculating the endogenous amount of the at least one glycan in the sample wherein the endogenous amount of the at least one glycan in the sample=the total amount of the at least one glycan in the sample−the amount of the at least one glycan added in b1).   
     
     
         16 . A method for diagnosing a disease or condition in a subject, the method comprising:
 (a) comparing the N-linked glycan profile from a subject to an N-linked glycan profile from a normal sample or diseased sample; and   (b) determining whether the subject has the disease or condition; wherein the glycan profile is determined by:   (i) obtaining a biological sample comprising at least one glycoprotein;   (ii) denaturing the at least one glycoprotein in the biological sample;   (iii) releasing at least one glycan from the at least one glycoprotein;   (iv) coating the biological sample with a matrix;   (v) analyzing the at least one glycan using mass spectrometry; and   
       wherein spatial distribution of the at least one glycan is maintained. 
     
     
         17 . The method of  claim 16 , wherein the disease or condition is cancer, an immunological disorder; a neurodegenerative disease, a transmissible spongiform encephalopathy, Alzheimer's disease, neuropathy, inflammation, rheumatoid arthritis, cystic fibrosis, or viral or bacterial infection. 
     
     
         18 . The method of  claim 16 , wherein the biological sample comprises a paraffin-embedded tissue. 
     
     
         19 . The method of  claim 16 , wherein the biological sample comprises a formalin-fixed tissue. 
     
     
         20 . The method of  claim 18 , wherein the biological sample is rehydrated. 
     
     
         21 . The method of  claim 16 , wherein the biological sample is deposited on a solid support. 
     
     
         22 . The method of  claim 21 , wherein the solid support allows sectioning so that spatial distribution is maintained. 
     
     
         23 . The method of  claim 16 , wherein denaturing the at least one glycoprotein comprises heating the biological sample and/or incubating the biological sample with a proteolytic enzyme for a sufficient period of time. 
     
     
         24 . The method of  claim 23 , wherein releasing the at least one glycan occurs by using an enzyme selected from the group consisting of Endoglycosidase H (Endo H), Endoglycosidase F (EndoF), and N-Glycanase F (PNGaseF). 
     
     
         25 . The method of  claim 24 , wherein releasing the at least one glycan occurs by using the enzyme PNGaseF. 
     
     
         26 . The method of  claim 25 , wherein releasing the at least one glycan occurs by using a microarray printer. 
     
     
         27 . The method of  claim 26 , wherein the matrix comprises 2,5-dihydroxybenzoic acid (DHB). 
     
     
         28 . The method of  claim 16 , wherein the mass spectrometry is MALDI-mass spectrometry. 
     
     
         29 . The method of  claim 28 , wherein the MALDI-mass spectrometry comprises MALDI time-of-flight (TOF) mass spectrometry. 
     
     
         30 . The method of  claim 29 , wherein the MALDI-mass spectrometry comprises MALDI-quadrupole ion trap (QIT)-TOF mass spectrometry. 
     
     
         31 . The method of  claim 16 , the method further comprising:
 b1) adding to the sample a known concentration of at least one glycoprotein;   e1) calculating the endogenous amount of the at least one glycan in the sample wherein the endogenous amount of the at least one glycan in the sample=the total amount of the at least one glycan in the sample−the amount of the at least one glycan added in b1).   
     
     
         32 . A method for improving the limit of detection of one or more target analytes in a sample, the method comprising:
 a) obtaining a sample;   b) adding to the sample a known concentration of one or more target analytes;   c) applying the sample to a matrix; and   d) analyzing the one or more target analytes using mass spectrometry,   
       wherein the limit of detection of the one or more target analytes is calculated using the formula Signal (C+LOD c )=Signal (C)+3SD c . 
     
     
         33 . A method for preserving and detecting sialic acid residues in a sample comprising:
 (a) obtaining a biological sample comprising at least one sialic acid containing glycoprotein;   (b) adding p-toluidine to the sample;   (c) denaturing the at least one sialic acid containing glycoprotein in the biological sample;   (d) releasing at least one sialic acid containing glycan from the at least one sialic acid containing glycoprotein;   (e) coating the biological sample with a matrix;   (f) analyzing the at least one sialic acid containing glycan using mass spectrometry; and   
       wherein spatial distribution of the at least one sialic acid containing glycan is maintained.