Protein UTLC-MALDI–MS using thin films of submicrometer silica particles

Abstract

Slides for ultra thin-layer chromatography (UTLC) slides were made by coating nonporous silica particles, chemically modified with polyacrylamide, as 15 μm films on glass or silicon. Three proteins, myoglobin, cytochrome c and lysozyme, are nearly baseline resolved by the mechanism of hydrophilic interaction chromatography. A plate height as low as 3 μm, with 3,900 plates, are observed in 14 mm. Varying silica particle diameter among 900, 700 and 350 nm showed that decreasing particle diameter slightly improves resolution but slows the separation. Matrix-assisted laser desorption/ionization (MALDI)-MS of the proteins after separation is demonstrated by wicking sufficient sinapinic acid into the separation medium.

Polyacrylamide brush layer for hydrophilic interaction liquid chromatography of intact glycoproteins

“…The silica particles were modified as described earlier [21-22]. Freshly rehydrolyzed silica particles were suspended in a dry toluene solution containing 2% (v/v) of (chloromethyl)phenylethyl-trichlorosilane and trichloromethylsilane at a volume ratio of 1:20.…”

“…The concept is depicted in Figure 1, where parent proteins would be separated by virtue of differential adsorption just at the PAAm surface, whereas carbohydrates might be separated by more subtle differences in structure by virtue of differential partitioning into the hydrophilic layer. Thin-layer chromatography (TLC) of proteins, albeit nonglycosylated, show that HILIC can be carried out with this bonded phase [21]. The purpose of this paper to investigate HILIC using the PAAm brush layer for Ultrahigh Performance Liquid Chromatography (UHPLC) of intact glycoproteins.…”

Recent advances in the preparation of adsorbent layers for thin‐layer chromatography combined with matrix‐assisted laser desorption/ionization mass‐spectrometric detection

“…MALDI mass spectra obtained from the spots of each of the three proteins after UTLC separation: (A) myoglobin, (B) cytochrome c, and (C) lysozyme …”

“…A set of six laser dyes, as well as a mixture of three labeled amino acids: lysine, threonine, and phenylalanine, were fully resolved on the carbon UTLC plate demonstrating tunable retention and plate number values above 10 000 theoretical plates. Later, other polymeric nanofibers (polyacrylonitrile, poly(vinyl alcohol) and carbon nanofibers [13]) were pyrolyzed to final glassy carbon ultra-thin layers demonstrating ability for surface-assisted laser desorption/ionization and F I G U R E 1 MALDI mass spectra obtained from the spots of each of the three proteins after UTLC separation: (A) myoglobin, (B) cytochrome c, and (C) lysozyme [14] matrix-enhanced surface-assisted laser desorption/ionization analyses which are discussed in Section 3.2.…”

Simple thin layer chromatography–ultraviolet spectrophotometric method for quality assessment of binary fixed‐dose‐combinations of lamivudine/tenofovir disoproxil fumarate and lamivudine/zidovudine in tablet formulations

“…TLC provides the most simple, rapid, and cost‐effective method with low volume consumption of solvents and many samples could be run simultaneously on a plate [17,26]. Also, in recent times TLC has been hyphenated to other detection devices or instruments, such as TLC–MS [22], UTLC–MALDI–MS [27], HPTLC–UV [22], and TLC–MALDI–TOF‐MS [28]. Kucherenko et al.…”