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We developed a simple, paper‐based device that enables sensitive detection by mass spectrometry (MS) without solid phase extraction or other sample preparation. Using glass fiber filter papers within a 3D printed holder, the device employs electrokinetic manipulations to stack, separate, and desalt charged molecules on paper prior to spray into the MS. Due to counter‐balanced electroosmotic flow and electrophoresis, charged analytes stack on the paper and desalting occurs in minutes. One end of the paper strip was cut into a sharp point and positioned near the inlet of a MS. The stacked analyte bands move toward the paper tip with the EOF where they are ionized by paper spray. The device was applied to analysis of PFAS in tap water with sub part‐per‐trillion detection limits in less than ten minutes with no sample pretreatment. Analysis of opioids in urine also occurs in minutes. The crucial parameters to enable stacking, separation, and MS ionization of both positively and negatively charged analytes were determined and optimized. Experimental and computational modeling studies confirm the electrokinetic stacking and analyte transport mechanisms. On‐paper separations were carried out by stacking analyte bands at different locations depending on their electrophoretic mobility, achieving baseline separation in some cases. [ABSTRACT FROM AUTHOR]

Sequential adsorption of poly(styrene sulfonate) and trypsin in nylon membranes provides a simple, inexpensive method to create stable, microporous reactors for fast protein digestion. The high local trypsin concentration and short radial diffusion distances in membrane pores facilitate proteolysis in residence times of a few seconds, and the minimal pressure drop across the thin membranes allows their use in syringe filters. Membrane digestion and subsequent MS analysis of bovine serum albumin provide 84% sequence coverage, which is higher than the 71% coverage obtained with in-solution digestion for 16 h or the 10.1021/ac101857j

Matrix-assisted laser desorption/ionization plates coated with poly(2-hydroxyethyl methacrylate) (PHEMA) brushes that are derivatized with Fe(III)-nitrilotriacetate (NTA) complexes allow selective, efficient phosphopeptide enrichment prior to analysis by mass spectrometry (MS). Fe(III)-NTA-PHEMA brushes (60 nm thick) have a phosphopeptide binding capacity of 0.6 [micro]g/[cm.sup.2] and exhibit phosphopeptide recoveries of over 70%, whereas much thinner polymer films containing Fe(III)-NTA afford a recovery of only 20%, and a monolayer of Fe(III)-NTA shows a recovery of just 10%. Recoveries are determined by comparing signals from enriched unlabeled phosphopeptides with those of their deuterium-labeled analogues that were added to the plate just prior to addition of matrix. Mass spectra of phosphopeptide-containing samples enriched using Fe(III)-NTA-PHEMA-modified plates also demonstrate higher recoveries or fewer interfering peaks than corresponding spectra obtained with enrichment using several commercially available Fe(III)-containing films and resins or metal oxide materials. When analyzing tryptic digests of [beta]-casein, the Fe(III)-NTA-PHEMA brushes allow detection of as little as 15 fmol of phosphopeptide. Moreover, with both ovalbumin and [beta]-casein digests, phosphopeptide signals dominate the mass spectra obtained using these modified plates.

Metal affinity complexes were chemically grafted onto the surface of gold matrix-assisted laser desorption/ionization (MALDI) plates by coupling a derivative of nitrilotdacetate (NTA) to immobilized poly(acrylic acid) (PAA) and subsequently forming the Fe(III)--NTA complex. The immobilized complexes can adsorb phosphorylated peptides preferentially from protein digests; deposition of digests on these surface-modified plates, followed by rinsing with an acetic acid solution, addition of matrix, and subsequent analysis by MALDI MS, resulted in mass spectra dominated by peaks corresponding to phosphopeptides. In the case of analyzing a tryptic digest of [beta]-casein, conventional MALDI MS revealed only one monophosphopeptide, while use of the Fe(III)--NTA--PAA-modified plate resulted in strong signals due to two additional tetraphosphorylated species. The diminution or elimination of signals due to nonphosphorylated species also greatly simplified the identification of phosphopeptides during analysis of ovalbumin digests and myoglobin digests spiked with an equimolar mixture of angiotensin and phosphoangiotensin. The matrix 2',4',6'-trihydroxyacetophenone mixed with diammonium hydrogen citrate proved to be much better than [alpha]-cyano-4-hydroxycinnamic acid for the detection of phosphorylated peptides from digests of [beta]-casein and ovalbumin.

Coating of substrates with polyelectrolyte multilayers terminated with poly(acrylic acid) (PAA) followed by activation of the free -COOH groups of PAA provides a surface that readily reacts with amine groups to allow covalent immobilization of antibodies. The use of this procedure to prepare arrays of antibodies in porous alumina supports facilitates construction of a flow-through system for analysis of fluorescenfly labeled antigens. Detection limits in the analysis of Cy5-labeled IgG are 0.02 ng/mL because of the high surface area of the alumina membrane, and the minimal diameter of the substrate pores results in binding limited by kinetics, not mass transport. Moreover, PAA-terminated films resist nonspecific protein adsorption, so blocking of antibody arrays with bovine serum albumin is not necessary. These microarrays are capable of effective analysis in 10% fetal bovine serum.

The use of sample probe surfaces patterned with 200[micro]m-diameter spots of hydrophilic, charged polymers significantly enhances the analysis of protein digests and DNA by MALDI-MS. Selective adsorption on these polymer-modified surfaces allows collection of specific proteolytic peptides, while subsequent rinsing of the deposited sample removes contaminants. In the case of partially digested myoglobin, the mass spectrum obtained using a sample probe modified with polyanionic functionalities permits detection of 22 proteolytic fragments, while analysis using a stainless steel MALDI sample probe gives only 11 detectable fragments. Similarly, during the analysis of bovine serum albumin digests, the use of several different surface-modified MALDI sample probes increases sequence coverage from 61.3 to 74.5%. Detection of phosphorylated peptides can be quite challenging during analyses of phosphoprotein digests by MALDI-MS because these anionic proteolytic fragments have low ionization efficiencies. However, MALDI signals from the phosphorylated proteolytic fragments sometimes increase dramatically when using a sample probe surface modified by a polycation (polyethylenimine or poly(acrylic acid) complexed with [Fe.sup.3+]). The signal enhancement apparently occurs because the positive surface selectively binds the phosphorylated peptides. The use of patterned, polycationic surfaces also shows great promise for selective adsorption and decontamination of DNA samples; a simple water rinse diminishes or eliminates the formation of multi-ion adducts, thereby improving mass resolution during subsequent analysis by MALDI-MS.

This paper describes a surface science/mass spectrometry effort to develop and characterize a patterned gold surface that serves as a MALDI sample platform capable of concentrating and purifying proteins. Using microcontact printing, small (200-[micro]m diameter) hydrophilic spots of bare gold or chemically anchored poly(acrylic acid) (PAA) are patterned at 5-mm intervals in a hydrophobic field consisting of a serf-assembled monolayer of hexadecanethiol. Building on recent innovations by others, the small hydrophilic spots concentrate the sample to achieve good reproducibility and high sensitivity in the MALDI signal. One of the key features in this work is the combination of the high density of carboxylate groups in PAA with a small spot size to afford both concentration and purification of proteins via ionic interactions. This translates into detection limits for salt-contaminated proteins that are 20-100 times lower (low femtomole) than those reported for previous polymer- or monolayer-modified MALDI probes (using proteins in the 3-15-kDa range). Reflectance Fr-IR spectroscopy and ellipsometry were used to determine the amount of protein adsorbed to a PAA-modified sample plate as a function of pH and salt concentration. Amide absorbances in IR spectra correlate well with MALDI-MS signals measured after addition of 2,5-dihydroxybenzoic acid as a matrix.

Ultrathin hyperbranched poly(acrylic acid) membranes were fabricated.