Mass spectrometry (MS) is the leading technique for rapid identification of proteins and characterization of their posttranslational modifications. However, as with most analyses, the success of MS methods depends critically on sample purification and pretreatment. Microporous membranes are attractive as a platform for enrichment and modification of proteins and peptides because convection and short radial diffusion distances in pores provide rapid analyte mass transport to binding or catalytic sites. Additionally, variation of the transmembrane flow rate can control the extent of reactions such as digestion. This research exploits facile layer-by-layer adsorption to incorporate nanoparticles or enzymes in micropores and create functional membranes. As tools for sample preparation prior to MS analysis, TiO2-modified membranes enrich phosphopeptides and protease-containing membranes afford control over protein digestion.Posttranslational protein phosphorylation is one of the most important mechanisms for creating protein diversity in eukaryotic cells. However due to the relatively low abundance of most phosphopeptides in proteolytic digests, phosphopeptide enrichment is a prerequisite step for effective and efficient MS analysis of phosphorylation. Alternating adsorption of poly(styrene sulfonate) (PSS) and TiO2 nanoparticles in porous nylon substrates creates TiO2-modified membranes for rapid enriching of phosphopeptides from small-volume (10's of &muL) samples. These TiO2-modified membranes bind 540 nmols of phosphopeptides in a 22-mm diameter disc and selectively isolate the phosphopeptides from -casein, -casein and ovalbumin digests even in a 100-fold excess of non-phosphorylated BSA digests if 2 % TFA is the loading and washing buffer. The combination of membrane enrichment and tandem MS reveals seven phosphorylation sites from in vivo phosphorylated tau protein, which is associated with Alzheimer's disease, and identification of such sites might aid the design of therapeutic drugs that inhibit phosphorylation.Using a similar approach to membrane modification, sequential adsorption of PSS and proteases in porous nylon substrates yields enzymatic membrane reactors for limited protein digestion. Although a high local enzyme density (~30 mg/cm3) and small pore diameters in the membrane lead to digestion in < 1 s, the low membrane thickness (170 &mum) affords control over residence times at the ms level to limit digestion. Most importantly, pepsin-containing membranes afford control of peptides sizes by altering the flow rate. Apomyoglobin digestion demonstrates that peptide lengths increase as the residence time in the membrane decreases. Under denaturing conditions, limited membrane digestion of bovine serum albumin and subsequent ESI-Orbitrap MS analysis reveal large peptides (3-10 kD) that increase the sequence coverage from 53 % (2-s digestion) to 82 % (0.05-s digestion). Moreover, electron transfer dissociation tandem MS on a large myoglobin proteolytic peptide (8 kD) provides a resolution of 1-2 amino acids, which suggests that this protease-modified membrane might find applications in hydrogen-deuterium exchange or middle-down proteomics studies. In addition, using trypsin- and &alpha-chymotrypsin-modified membranes, a limited proteolysis study of a large Arabidopsis GTPase, ROOT HAIR DEFECTIVE 3, shows suitable probing for labile regions near the C-terminus to suggest what protein reconstruction might make RHD3 more suitable for crystallization. These studies clearly demonstrate the potential of functional membranes for rapid and controlled purification or pretreatment prior to MS analysis.
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