Improving Proteome Coverage by Reducing Sample Complexity via Chromatography.

High performance liquid chromatography (HPLC) is currently one of the most powerful analytical tools that has revolutionized the field of proteomics. Formerly known as high pressure liquid chromatography, this technique was introduced in the early 1960s to improve the efficiency of liquid chromatography separations using small stationary phase particles packed in columns. Since its introduction, continued advancements in column technology, development of different stationary phase materials and improved instrumentation has allowed the full potential of this technique to be realized. The various modes of HPLC in combination with mass spectrometry has evolved into the principal analytical technique in proteomics. It is now common practice to combine different types of HPLC in a multidimensional workflow to identify and quantify peptides and proteins with high sensitivity and resolution from limited amounts of samples. More recently, the introduction of Ultra High Performance Liquid Chromatography (UHPLC) has further raised the level of performance of this technique with significant increases in resolution, speed and sensitivity. The number of applications of HPLC and UHPLC in proteomics has been rapidly expanding and will continue to be a pivotal analytical technique. The aim of the following sections is to familiarize the beginner with the various HPLC methods routinely used in proteomics and provide sufficient practical knowledge regarding each of them to develop a separation and analytical protocol.

On-target and nanoparticle-facilitated selective enrichment of peptides and proteins for analysis by MALDI-MS.

Over the course of the last decade, a number of investigators have come to appreciate that the surface of a MALDI target, after suitable modification, can be used for selective enrichment of peptides and proteins. More recently, surface-modified nanoparticles (NPs) that readily co-crystallize in MALDI matrix, are not ionized by laser desorption/ionization, and do not interfere with MS have attracted interest as alternatives to surface-modified targets for selective enrichment of peptides and proteins. Surface-modified targets and NPs facilitate parallel processing of samples, and when used in conjunction with MALDI mass spectrometers with kHz lasers enable development of high-throughput proteomics platforms. Targets and NPs for reversed phase and ion exchange retention, selective enrichment of glycopeptides, selective enrichment of phosphopeptides, and immunoaffinity MS are described in conjunction with details regarding their preparation and utility. Commercial availability of the reagents and substrates required to prepare surface-modified targets and NPs is also discussed.

Biochips that sequentially capture and focus antigens for immunoaffinity MALDI-TOF MS: a new tool for biomarker verification.

A novel approach to immunoaffinity MS is described wherein antibodies are appended to a patterned gold Biochip surface. The Biochip surface is patterned with an array of concentric immunocapture zones composed of highly hydrophilic central zones surrounded by moderately hydrophilic zones that reside on a non-wetting background, with protein attachment via electrochemically cleavable linkers. After linker cleavage, matrix application forms a discrete spot suitable for MALDI-TOF-MS. Use of the Biochip to purify transthyretin from human serum allowed a distinct resolution of four disulfide conjugates and one truncated form isoforms with good mass resolution and sensitivity.

Use of fluorescent sequence-specific polyamides to discriminate human chromosomes by microscopy and flow cytometry.

In this paper, we demonstrate the use of synthetic polyamide probes to fluorescently label heterochromatic regions on human chromosomes for discrimination in cytogenetic preparations and by flow cytometry. Polyamides bind to the minor groove of DNA in a sequence-specific manner. Unlike conventional sequence-specific DNA or RNA probes, polyamides can recognize their target sequence without the need to subject chromosomes to harsh denaturing conditions. For this study, we designed and synthesized a polyamide to target the TTCCA-motif repeated in the heterochromatic regions of chromosome 9, Y and 1. We demonstrate that the fluorescently labeled polyamide binds to its target sequence in both conventional cytogenetic preparations of metaphase chromosomes and suspended chromosomes without denaturation. Chromosomes 9 and Y can be discriminated and purified by flow sorting on the basis of polyamide binding and Hoechst 33258 staining. We generate chromosome 9- and Y-specific 'paints' from the sorted fractions. We demonstrate the utility of this technology by characterizing the sequence of an olfactory receptor gene that is duplicated on multiple chromosomes. By separating chromosome 9 from chromosomes 10-12 on the basis of polyamide fluorescence, we determine and differentiate the haplotypes of the highly similar copies of this gene on chromosomes 9 and 11.