Methods for early detection of blood disorders
Disclosed herein are methods of detecting blood disorders, such as diabetes. In particular examples the method includes contacting a blood sample with an amine reagent, blocking an excess of the amine reagent with a blocking reagent, digesting the modified blood sample with trypsin to produce a digested blood sample containing a plurality of glycated N-terminal peptides and non-glycated N-terminal peptides, then analyzing the digested blood sample with MALDI MS. Also provided are reagents for use in such methods.
1. A method for detecting a blood disorder in a subject, comprising:
contacting a blood sample obtained from a subject with an amine reactive reagent, thereby modifying said blood sample;
blocking any of the amine reactive reagent in the modified blood sample with a blocking reagent selected therefor;
subsequently digesting the modified blood sample with a trypsin composition, thereby producing a digested blood sample comprising a first plurality of glycated N-terminal peptides and a second plurality of non-glycated N-terminal peptides;
analyzing said digested blood sample with MALDI MS to detect therein and separate therefrom the first plurality of glycated N-terminal peptides and the second plurality of non-glycated N-terminal peptides;
comparing and correlating said first plurality of glycated N-terminal peptides and the second plurality of non-glycated N-terminal peptides with at least one selected reference data set; and
determining therefrom said comparison and correlation the presence or absence of the blood disorder.
2. The method according to claim 1 , wherein the first plurality of N-terminal peptides comprise at least one of albumin, α-hemoglobin, and β-hemoglobin.
3. The method according to claim 1 , wherein said blood sample is a dried blood sample.
4. The method according to claim 3 , wherein said dried blood sample is storable.
5. The method according to claim 1 , where said blood sample is a liquid blood sample.
6. The method according to claim 5 , wherein said liquid blood sample is storable.
7. The method according to claim 1 , wherein a detection of elevated glycated β-hemoglobin in comparison to the reference data set is an indicator of a diabetes condition.
8. The method according to claim 1 , wherein said blood sample is a whole blood sample.
9. The method according to claim 1 , wherein said amine reactive reagent modifies one of a plurality of N-terminals and a plurality of lysine side chains of protein components in the blood sample.
10. The method according to claim 1 , wherein said blood sample is a fractionated blood sample.
11. The method according to claim 1 , wherein the amine reactive reagent comprises a general formula of R1—R2 wherein R1 is a reactive group specific for modification of primary amino groups.
12. The method according to claim 11 , wherein the R1 group is selected from the group consisting of an N-hydroxysuccinimidyl ester, N-hydroxysulfosuccinimidyl ester, isothiocyanate, pentafluorophenyl ester, sulfotetrafluorophenyl ester, sulfonyl chloride, p-nitrophenyl, aldehyde, and combinations thereof.
13. The method according to claim 11 , wherein the R2 group is selected from the group consisting of a pyridinyl, piperidinyl, N-alkylpiperidinyl, piperazinyl, N-alkylpiperazinyl, imidazolyl, N-alkylimidazolyl, dialkylamine, trialkylamine, and combinations thereof.
14. The method of claim 1 , wherein comparing and correlating comprises quantifying the first plurality of glycated N-terminal peptides and quantifying the second plurality of non-glycated N-terminal peptides.
15. The method of claim 1 , wherein comparing and correlating comprises determining a percent of glycation of the N-terminal peptides.
16. The method of claim 1 , wherein the blood disorder is sickle cell, hemoglobin C, hemoglobin S-C, hemoglobin E, thalassemia or analbuminaemia.
17. The method of claim 1 , wherein the blood disorder is associated with type I or type II diabetes or a mutated hemoglobin.