SRM METHODS IN ALZHEIMER'S DISEASE AND NEUROLOGICAL DISEASE 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 claim 7 , wherein each prioritized set of peptides contains 1-5 peptides.

10 . The assay according to claim 1 , 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 . An assay developed according to the method of claim 1 .