Wisconsin Alumni Research Foundation

University of Illinois

Neil L Kelleher

Lloyd M Smith

Joshua J Coon

Madhusudan Patel

Peter Jeremy Belshaw

Brian L Frey

Michael Robert Shortreed

Casey J Krusemark

Margaret F Phillips

Shane M Lamos

Gregory K Taylor

Relative quantification of metabolites by Electrospray Ionization Mass Spectrometry (ESI-MS) requiring a mechanism for simultaneous analysis of multiple analytes in two or more samples. Labeling reagents that are reactive to particular compound classes and differ only in their isotopic compositions facilitate relative quantification. Heavy and light isotopic forms of methylacetimidate were synthesized and used as labeling reagents for quantification of amine-containing molecules. Heavy and light isotopic forms of formaldehyde and cholamine were also synthesized and used independently as labeling reagents for quantification of amine-containing and carboxylic acid-containing molecules, such as found in biological samples. The labeled end-products are positively charged under normal acidic conditions involving conventional Liquid Chromatography Mass Spectrometry (LC/MS) applications. Labeled primary and secondary amine and carboxylic acid end-products generated higher signals concerning mass-spectra than pre-cursor molecules and improved sensitivity. Improved accuracy concerning relative quantification was demonstrated by mixing heavy and light labeledextracts in different ratios.

Ionizable isotopic labeling reagents for relative quantification by mass spectrometry

Relative quantification of metabolites by Electrospray Ionization Mass Spectrometry (ESI-MS) requiring a mechanism for simultaneous analysis of multiple analytes in two or more samples. Labeling reagents that are reactive to particular compound classes and differ only in their isotopic kit facilitating relative quantification and providing tangible evidence for the existence of specific functional groups. Heavy and light isotopic forms of methylacetimidate were synthesized and used as labeling reagents for quantification of amine-containing molecules, such as biological samples. Heavy and light isotopic forms of formaldehyde and cholamine were also synthesized and used independently as labeling reagents for quantification of amine-containing and carboxylic acid-containing molecules, such as found in biological samples. Advantageously, the labeled end-products are positively charged under normal acidic conditions involving conventional Liquid Chromatography Mass Spectrometry (LC/MS) applications. Labeled primary and secondary amine and carboxylic acid end-products also generated higher signals concerning mass-spectra than pre-cursor molecules and improved sensitivity. Improved accuracy concerning relative quantification was achieved by mixing heavy and light labeledextracts in different ratios. Labeling strategy was further employed to ascertain differences in the amounts of amine-containing metabolites for two strains ofseeds.

A method of preparing a first set of candidate fragments from a sample protein fragment and a protein sequence, comprises selecting a first candidate sequence comprising a terminal amino acid of the protein sequence; generating a further candidate sequence from each candidate sequence, except a last candidate sequence; and including any candidate sequences having a mass which is equal to the mass of the sample protein fragment within a third tolerance, in the first set of candidate fragments. The generating of the further candidate sequences from each candidate sequence is by adding a portion of the protein sequence farther away from the terminal amino acid than the candidate sequence, if a mass of the sample protein fragment is equal to or greater than the mass of the candidate sequence within a first tolerance, or deleting a portion of the candidate sequence from an end closest to the terminal amino acid, if the mass of the sample protein fragment is less than the mass of the candidate sequence within a second tolerance. The candidate sequences are subsequences of the protein sequence.

Identification and characterization of protein fragments

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Urbana, IL, United States of America

Neil L Kelleher