Srm methods in alzheimer's disease and neurological disease assays

Abstract

Provided herein are methods for developing selected reaction monitoring mass spectrometry (LC-SRM-MS) assays.

1 . A multiplexed LC-SRM-MS assay for the measurement of a plurality of proteins in a single sample comprising:
 a) generating a set of optimal peptides and corresponding transitions for each protein monitored;   b) optimizing the collision energy for each transition such that interference among the transitions monitored is avoided;   c) selecting a set of transitions that have the greatest peak areas are monitored for each of the proteins, and wherein the selected transitions do not interfere with the ions in the sample;   d) monitoring the detected set of transitions for each protein in the sample, thereby measuring a plurality of proteins in the sample.   
     
     
         2 . The assay of  claim 1 , wherein each monitored peptide
 (i) has a monoisotopic mass of 700-5000 Da; and   (ii) does not contain a cysteine or a methionine; and.   
     
     
         3 . The assay of  claim 1 , wherein the transitions for each peptide
 (i) have one of the four most intense b or y transition ions;   (ii) has m/z values of at least 30 m/z above or below those of a precursor ion;   (iii) do not interfere with transitions from other peptides; and   (iv) represent transitions due to breakage of peptide bond at different sites of the protein.   
     
     
         4 . The assay according to  claim 1 , wherein the peptides do not include any peptide that is bounded by KK, KR, RK or RR, either upstream of downstream in the corresponding protein sequence. 
     
     
         5 . The assay according to  claim 1 , wherein each peptide of said set of peptides is unique to the corresponding protein. 
     
     
         6 . The assay according to  claim 1 , wherein the peptides do not include peptides which were observed in post-translational modified forms. 
     
     
         7 . The assay according to  claim 1 , wherein each set of peptides is prioritized according to one or more of the following ordered set of criteria:
 (a) unique peptides first, then non-unique;   (b) peptides with no observed post-translational modifications first, then those observed with post-translational modifications;   (c) peptides within the mass range 800-3500 Da first, then those outside of 800-3500 Da; and   (d) sorted by decreasing number of variant residues.   
     
     
         8 . The assay according to  claim 7 , wherein each set of peptides is prioritized according to all of the ordered set of criteria. 
     
     
         9 . The assay according to any one of  claims 7  or  8 , wherein each prioritized set of peptides contains 1-5 peptides. 
     
     
         10 . The assay according to any one of  claims 1 - 9 , wherein the two best peptides per protein and the two best transitions per peptide are selected based on experimental data resulting from LC-SRM-MS analysis of one or more of the following experimental samples: a biological disease sample, a biological control sample, and a mixture of synthetic peptides of interest. 
     
     
         11 . The assay according to  claim 10 , wherein the biological disease and biological control samples are processed using an immunodepletion method prior to LC-SRM-MS analysis. 
     
     
         12 . The assay according to  claim 11 , wherein the experimental samples contain internal standard peptides. 
     
     
         13 . The assay according to  claim 11 , wherein the LC-SRM-MS analysis method specifies a maximum of 7000 transitions, including transitions of the internal standard peptides and transitions. 
     
     
         14 . The assay according to  claim 1 , wherein the top two transitions per peptide are selected according to one or more of the following criteria:
 (1) the transitions exhibit the largest peak areas measured in either of the two biological experimental samples;   (2) the transitions are not interfered with by other ions;   (3) the transitions do not exhibit an elution profile that visually differs from those of other transitions of the same peptide;   (4) the transitions are not beyond the detection limit of both of the two biological experimental samples; and   (5) the transitions do not exhibit interferences.   
     
     
         15 . The assay according to  claim 1 , wherein the top two peptides per protein are selected according to one or more of the following criteria:
 (1) one or more peptides exhibit two transitions according to  claim 12  and represent the largest combined peak areas of the two transitions according to  claims 12 ; and   (2) one or more peptides exhibit one transition according to  claim 12  and represent the largest combined peak areas of the two transitions according to  claim 12 .   
     
     
         16 . An assay developed according to the method of  claim 1 .