Methods for assaying palmitoyl protein thioesterase 1 and tripeptidyl peptidase activity in dried blood spots for detection of neuronal ceroid lipofuscinoses in newborns

A method for assaying for a lysosomal enzyme associated with a lysosomal storage disease, comprising:

• (a) contacting a sample with an aqueous solution to provide a solution comprising one or more lysosomal enzymes;
• (b) contacting the one or more lysosomal enzymes in solution with one or more lysosomal enzyme substrates, and incubating the substrates with the lysosomal enzymes for a time sufficient to provide a solution comprising one or more enzyme products for the one or more lysosomal enzymes present in the sample,
• wherein the enzyme substrate comprises a compound of Formula (TPP1-S1) and/or Formula (PPT1-S1):
• wherein in the compound of Formula (TPP1-S1),
• P3 is an achiral amino acid residue, a D-amino acid residue, or a L-amino acid residue;
• P2 is an amino acid residue selected from D-Nle, L-Nle, D-Pro, L-Pro, D-Ala, L-Ala, D-Phe, L-Phe, D-Tyr, L-Tyr, D-Trp, L-Trp, D-Ile, L-Ile, D-Val, L-Val, D-Thr, L-Thr, D-Glu, L-Glu, D-Ser, L-Ser, D-Leu, L-Leu, Gly, D-Gln, L-Gln, D-Lys, L-Lys, D-Asn, L-Asn, D-Asp, L-Asp, D-Arg, L-Arg, D-His, and L-His;
• P1 is an amino acid residue selected from D-Phe, L-Phe, D-Leu, L-Leu, D-Nle, L-Nle, D-Tyr, L-Tyr, D-Glu, L-Glu, D-Trp, L-Trp, D-Asp, L-Asp, D-Gln, L-Gln, D-Ala, L-Ala, D-Ser, L-Ser, D-Lys, L-Lys, D-Arg, L-Arg, D-Asn, L-Asn, D-His, L-His, D-Val, L-Val, D-Ile, L-Ile, D-Thr, L-Thr, Gly, D-Pro, and L-Pro;
• wherein P3 is located at a N-terminus of the P3-P2-P1 moiety, P1 is located at a C-terminus of the P3-P2-P1 moiety;
• G1 is a first linker comprising 2-20 carbon atoms in which one or more carbon atoms may be replaced with a heteroatom selected from N, O, and S, and/or one or more of carbon atoms may be substituted with a C1-C6 alkyl group, C1-C6 haloalkyl, halo, or amino;
• XA is a first linking functionality linking G1 to G2 selected from a bond, amide, thioamide, sulfonamide, ether, sulfide, ureido, thioureido, and ester;
• G2 is a second linker comprising 2-20 carbon atoms in which one or more carbon atoms may be replaced with a heteroatom selected from N, O, and S, and/or one or more of carbon atoms may be substituted with a C1-C6 alkyl group or halogen;
• and
• XB is a second linking functionality linking G2 to Prot selected from a bond, NH, amide, thioamide, sulfonamide, ether, sulfide, ureido, thioureido, and ester;
• and
• Prot is a protecting group that is capable of fragmenting by one dominant pathway at an efficiency of greater than 5% to provide a residual enzyme product fragment that is detectable by mass spectrometry;
• and
• wherein in the compound of Formula (PPT1-S1),
• RA is selected from a peptide, a polymer, a polysaccharide, an aromatic hydrocarbon group, and an aliphatic hydrocarbon group;
• W is selected from H and NHCO—C1-C6 alkyl;
• and
• Y is a C2-C19 alkylene;
• and
• (c) determining the quantities of the one or more enzyme products.