Enhanced sample multiplexing for nitrotyrosine-modified proteins using combined precursor isotopic labeling and...

Area of Science:

• Proteomics
• Biochemistry
• Analytical Chemistry

Background:

• Current methods for identifying and quantifying 3-nitrotyrosine (3NT) post-translationally modified proteins (PTM) typically involve biotin/avidin enrichment.
• Existing quantitative techniques utilize isotopic labeling or isobaric tagging for relative quantification of 3NT-modified proteins in limited sample sets (2 or 8 samples).

Purpose of the Study:

• To introduce a novel strategy, combined precursor isotopic labeling and isobaric tagging (cPILOT), for enhanced multiplexing in quantifying 3NT-PTMs.
• To increase the number of samples that can be analyzed for 3NT-PTMs using commercially available tandem mass tags (TMT) or isobaric tags for relative and absolute quantification (iTRAQ) reagents.

Main Methods:

• The cPILOT strategy involves blocking N-termini and lysine residues of tryptic peptides using light and heavy labeled acetyl groups.
• 3-nitrotyrosine (3NT) is reduced to 3-aminotyrosine (3AT) using sodium dithionite.
• Light and heavy labeled 3AT-peptides are subsequently derivatized with TMT or iTRAQ multiplex reagents.

Main Results:

• The study demonstrates the proof-of-principle utility of cPILOT.
• Experiments were conducted using in vitro nitrated bovine serum albumin (BSA) and mouse splenic proteins.
• The method was validated using TMT(0), TMT(6), and iTRAQ(8) reagents, showcasing its applicability.

Conclusions:

• cPILOT significantly expands the multiplexing capacity for quantifying 3NT-modified proteins, enabling analysis of up to 12 or 16 samples.
• The developed strategy offers a more comprehensive approach to studying 3NT-PTMs in complex biological samples.
• Limitations of the cPILOT strategy are discussed, paving the way for future refinements.