Characterization of Prenylated C-terminal Peptides Using a Thiopropyl-based Capture Technique and LC-MS/MS.

Posttranslational modifications play a critical and diverse role in regulating cellular activities. Despite their fundamentally important role in cellular function, there has been no report to date of an effective generalized approach to the targeting, extraction, and characterization of the critical c-terminal regions of natively prenylated proteins. Various chemical modification and metabolic labeling strategies in cell culture have been reported.

Multistep mass tagging coupled with 2D LC-MS--an approach to increasing the number of identified proteins.

Identification of large numbers of proteins from complex biological samples is a continuing challenge in the area of quantitative proteomics. We introduce here a simple and reliable multistep mass tagging technique using our recently developed solid phase mass tagging reagents. When coupled with two-dimensional liquid chromatography/nano-electrospray ionization ion trap mass spectrometry (2D-LC/nano-ESI-MS), this method allows enhanced protein identification when tested on samples from prokaryotic and eukaryotic sources.

Design and synthesis of a solid-phase fluorescent mass tag.

In this study, we demonstrate the design of a new solid-phase fluorescent mass tag (FMT) that contains the following features: (1) the FMT is synthesized using Fmoc chemistry which is simple, rapid, and cost-effective; (2) lysine is used as a uniformly labeled amino acid (using stable isotopes) to allow 8 Da difference between "heavy" and "light" tags; (3) a fluorescent molecule is coupled to the isotope tag that allows a tagged peptide to be detected by online fluorescence; and (4) an iodoacetyl reactive group provides cysteine reactivity. Using MALDI-TOF MS and HPLC, we show that the FMT reagent can be used to label standard cysteine-containing peptides as well as cysteine-containing peptides from a BSA tryptic digest.

Quantitative analysis of membrane proteins from breast cancer cell lines BT474 and MCF7 using multistep solid phase mass tagging and 2D LC/MS.

We introduce a new multistep mass tagging technique and show its utility for reducing sample complexity when coupled with two-dimensional liquid chromatography/nano-electrospray ionization ion trap mass spectrometry (2D LC/nano ESI-MS). Solid-phase mass tagging reagents were used to identify and obtain relative quantitation of membrane proteins from two established breast cancer cell lines, BT474 and MCF7. The results presented in this study show that sample complexity can be reduced with corresponding increases in protein identification and quantitation.

Incorporation of single-wall carbon nanotubes into an organic polymer monolithic stationary phase for mu-HPLC and capillary electrochromatography.

Single-wall carbon nanotubes (SWNT) were incorporated into an organic polymer monolith containing vinylbenzyl chloride (VBC) and ethylene dimethacrylate (EDMA) to form a novel monolithic stationary phase for high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). The retention behavior of neutral compounds on this poly(VBC-EDMA-SWNT) monolith was examined by separating a mixture of small organic molecules using micro-HPLC. The result indicated that incorporation of SWNT enhanced chromatographic retention of small neutral molecules in reversed-phase HPLC presumably because of their strongly hydrophobic characteristics.

2D LC/MS analysis of membrane proteins from breast cancer cell lines MCF7 and BT474.

Membrane proteins play a central role in the interaction of the cell with its environment and in the function of subcellular organelles. The current study focused on developing a better understanding of the membrane proteome of two well-characterized breast cancer cell lines. Membranes from osmotically lysed BT474 and MCF7 cells were treated with cyanogen bromide followed by a combination of trypsin and Staphylococcus V8 protease to obtain hydrophilic peptides from membrane proteins.

The role of liquid chromatography in proteomics.

Proteomics represents a significant challenge to separation scientists because of the diversity and complexity of proteins and peptides present in biological systems. Mass spectrometry as the central enabling technology in proteomics allows detection and identification of thousands of proteins and peptides in a single experiment. Liquid chromatography is recognized as an indispensable tool in proteomics research since it provides high-speed, high-resolution and high-sensitivity separation of macromolecules.

Capillary electrochromatography of peptides and proteins.

This paper reviews recent progress in bioanalysis using capillary electrochromatography (CEC), especially in the field of separation of proteins and peptides. Fundamentals of CEC are briefly discussed. Since most of the recent developments on CEC have focused on column technology, i.

A simple solid phase mass tagging approach for quantitative proteomics.

New mass-tagging reagents for quantitative proteomics measurements have been designed using solid phase peptide synthesis technology. The solid phase mass tags have been used to accurately measure the relative amounts of cysteine-containing peptides in model peptide mixtures as well as in mixtures of tryptic digests in the femtomol range. Measurements were made using both matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and online reversed-phase capillary liquid chromatography coupled through a nanoelectrospray interface to an ion trap mass spectrometer (capillary LC/ESI-MS).

Capillary electrochromatography of peptides on a neutral porous monolith with annular electroosmotic flow generation.

A new kind of monolithic capillary column was prepared for capillary electrochromatography (CEC) with a positively charged polymer layer on the inner wall of a fused-silica capillary and a neutral monolithic packing as the bulk stationary phase. The fused-silica capillary was first silanized with 3-glycidoxypropyltrimethoxysilane (GPTMS). Polyethyleneimine (PEI) was then covalently bonded to the GPTMS coating to form an annular positively charged polymer layer for the generation of electroosmotic flow (EOF).

Elution-modified displacement chromatography coupled with electrospray ionization-MS: on-line detection of trace peptides at low-femtomole level in peptide digests.

Elution-modified displacement chromatography (EMDC) was employed to achieve peptide separations with high efficiency. On-line ESI-MS and ESI-MS/MS measurements showed enrichment and detection of kemptide, a protein kinase A peptide substrate, at low femtomole levels when it was added as a trace marker component to a tryptic digest of bovine serum proteins or to a human growth hormone peptide digest at concentration ratios between 1:10(5) and 1:10(6). In another EMDC separation, five peptides were detected in a mixture containing 20 fmol of human growth hormone tryptic digest mixed with the bovine serum protein digest.

Selective enrichment of low-abundance peptides in complex mixtures by elution-modified displacement chromatography and their identification by electrospray ionization mass spectrometry.

Trace components were selectively enriched and detected in the tryptic digest of recombinant human growth hormone using elution-modified displacement chromatography, a hybrid technique combining features of elution and displacement chromatography. Based on the retention behavior of sample components in the elution mode, rapid and selective trace enrichment and high-resolution separation was achieved in a single step by utilizing appropriate combinations of an eluent such as aqueous acetonitrile with the displacer. Mass spectral and chromatographic analysis of displacement zones revealed up to 400-fold enhancement of the concentration of some low-abundance sample components.