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The study of protein phosphorylation has grown exponentially in recent years, as it became evident that important cellular functions are regulated by phosphorylation and dephosphorylation of proteins on serine, threonine and tyrosine residues. The use of immobilized metal affinity chromatography (IMAC) to enrich phosphopeptides from peptide mixtures has been shown to be useful especially prior to mass spectrometric analysis. For the selective enrichment applying solid-phase extraction (SPE) of phosphorylated peptides, we introduce poly(glycidyl methacrylate/divinylbenzene) (GMD) derivatized with imino-diacetic acid (IDA) and bound Fe(III) as a material. GMD is rapidly synthesized and the resulting free epoxy groups enable an easy access to further derivatization with, e.g., IDA. Electron microscopy showed that the synthesized GMD-IDA-Fe(III) for SPE has irregular agglomerates of spherical particles. Inductively coupled plasma (ICP) analysis resulted in a metal capacity of Fe(III) being 25.4 micromol/mL. To enable on-line preconcentration and desalting in one single step, GMD-IDA-Fe(III) and Silica C18 were united in one cartridge. Methyl esterification (ME) of free carboxyl groups was carried out to prevent binding of nonphosphorylated peptides to the IMAC function. The recovery for a standard phosphopeptide using this SPE method was determined to be 92%. The suitability of the established system for the selective enrichment and analysis of model proteins phosphorylated at different amino acid residues was evaluated stepwise. After successful enrichment of beta-casein deriving phosphopeptides, the established system was extended to the analysis of in vitro phosphorylated proteins, e.g. deriving from glutathione-S-transferase tagged extracellular signal regulated kinase 2 (GST-ERK2).

Over the past couple of years, proteomics pattern analysis has emerged as an effective method for the early diagnosis of diseases such as ovarian, breast, or prostate cancer, without identification of single biomarkers. MALDI-TOF MS, for example, offers a simple approach for fast and reliable protein profiling, especially by using carrier materials with various physical and chemical properties, in combination with a MALDI matrix. This approach is referred to as materialenhanced laser desorption/ionization (MELDI). In this paper, we report the development and application of derivatized carrier materials [cellulose, silica, poly(glycidyl methacrylate/divinylbenzene) (GMA/DVB) particles, and diamond powder] for fast and direct MALDI-TOF MS protein profiling. The applicability of MELDI for rapid protein profiling was evaluated with human serum samples. These carriers, having various hydrophobicities, resulted in characteristic mass fingerprints, even if all materials were derivatized with iminodiacetic acid (IDA) to yield an immobilized metal affinity chromatography (IMAC) functionality. Our study demonstrates that analyzing complex biological samples, such as human serum, by employing different MELDI carrier materials yielded type-and size-dependent performance variation. T he quest to diagnose and identify diseases at early stages depends mainly on the accurate determination of the marker entities in the biological fluids. These marker entities are normally very low concentrated [1], and highly sensitive methods are required to bind them effectively from the biosamples and to measure them with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) [2]. MALDI mass spectrometry, in recent times, has proven to be an authentic technique to analyze peptides and proteins [3,4]. These biomolecules out of the complex biofluids like serum are difficult to ionize and thus, bring into time of flight (TOF) because of the complexity of the sample and the suppressing nonvolatile salts [5]. The aforementioned problems are tackled by another technique introduced years ago, namely, surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF MS), for the detection of several peptides and proteins from complex biological specimens [6 -8]. This affinity-based method utilizes selective adsorption of proteins on various chromatographic surfaces to reduce sample complexity [9,10]. Spectra generated by SELDI-TOF MS usually show limited peak and binding capacity due to the smaller surface area of the plane protein chip [11]. Limitations of SELDI are also associated with the lower mass resolution of the applied MS instrument that

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Copoly(eugenol-DVB) with DVB composition of 2%, 6% and 12% had been prepared