Bifunctional polymer brushes for low-bias enrichment of mono- and multi-phosphorylated peptides prior to mass spectrometry analysis

Abstract

Polymer brushes orthogonally derivatized with oxotitanium and nitrilotriacetate-Fe(III) groups enrich both mono- and multi-phosphorylated peptides for mass spectrometry analysis.

Fig. 1

A. Scheme for orthogonal modification of PGMA brushes (see Fig. S1† for more details). B. PGMA-based control polymers (i and g). C. On-plate phosphopeptide enrichment for MALDI-MS analysis.

Fig. 2

Reflectance FTIR spectra of a PGMA film on a Au-coated wafer before (a) and after sequential reactions with the following reagents: (b) NaN₃; (c) SA; (d) N-hydroxysulfosuccinimide (sulfo-NHS)/N,N′-diisopropylcarbodiimide; (e) aminobutyl NTA; (f) propiolic acid (PA). The polymer abbreviation and ellipsometric thickness of the film (dry form) after each step are noted above each spectrum. The ESI† further explains the IR spectra.

Fig. 3

MALDI mass spectra after phosphopeptide enrichment from a 1-μL solution containing three digested phosphoproteins (3pp), α-casein, βcasein and ovalbumin, or a mixture of 3pp and BSA. The polymers employed for enrichment, their thicknesses (prior to addition of oxoTi or Fe(III)), and the sample acidity are noted above each spectrum. Each phosphopeptide is labeled as #p#, where the first # denotes the number of phosphatesin that peptide and the second # denotes the peptide number in Table S2†. Black and red labels represent mono- and multi-phosphorylated peptides, respectively.