Isobarically labeled analytes and fragment ions derived therefrom

A method for proteomic analysis of polypeptides, comprising:

• a) reacting each of two or more polypeptide samples, each sample comprising a reactive polypeptide analyte, with a different isobaric and/or isomeric labeling reagent of a set of isobaric and/or isomeric labeling reagents to thereby produce two or more differentially labeled polypeptide analytes having the same mass, wherein each labeling reagent is a 6 membered heterocyclic ring comprising a ring nitrogen atom that is N-alkylated with a substituted or unsubstituted acetic acid moiety to which the polypeptide analyte is linked through a carbonyl carbon of the N-alkyl acetic acid moiety
• b) mixing two or more of the differentially labeled polypeptide samples, or a portion thereof, and optionally one or more calibration standards to thereby produce a sample mixture comprising the same reactive polypeptide analyte labeled with two or more different labels having the same mass;
• c) digesting each differentially labeled polypeptide sample with at least one enzyme to degrade components of the sample, to form degraded samples, after performing step (a);
• and
• d) performing a mass analysis of the components of the degraded samples.