Biomedical subjects

Isabel Feuerstein

Publications and source records attributed to Isabel Feuerstein.

8 recordsLinked to original sources

PubMed2006

Material-enhanced laser desorption/ionization (MELDI)--a new protein profiling tool utilizing specific carrier materials for time of flight mass spectrometric analysis.

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 material-enhanced 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.

Indicators and Reagents↗

PubMed2006

Biomarker discovery in breast cancer serum using 2-D differential gel electrophoresis/ MALDI-TOF/TOF and data validation by routine clinical assays.

In the present study, we used 2-D differential gel electrophoresis (2-D DIGE) and MS to screen biomarker candidates in serum samples obtained from 39 patients with breast cancer and 35 controls. First, we pooled the serum samples matched with age and menopausal status. Then, we depleted the two most abundant proteins albumin and IgG by immunoaffinity chromatography under partly denaturing conditions in order to enrich low-abundance proteins and proteins with low molecular weight. Concentrated and desalted samples were labeled with three different CyDyes including one internal standard, pooled from all the samples, and separated with 2-D DIGE in triplicate experiments. Biological variations of the protein expression level were analyzed with DeCyder software and evaluated for reproducibility and statistical significance. The profile of differentially expressed protein spots between patients and controls revealed proapolipoprotein A-I, transferrin, and hemoglobin as up-regulated and three spots, apolipoprotein A-I, apolipoprotein C-III, and haptoglobin alpha2 as down-regulated in patients. Finally, routine clinical immunochemical reactions were used to validate selected candidate biomarkers by quantitative determination of specific proteins in all individual serum samples. The serum level of transferrin correlated well with the 2-D-DIGE results. However, the serum levels of apolipoprotein A-I and haptoglobin could not be detected with the clinical routine diagnostic tests. This demonstrated an advantage 2-D DIGE still has over other techniques. 2-D DIGE can distinguish between isoforms of proteins, where the overall immunochemical quantification does fail due to a lack of isoform-special antibodies.

Adult↗

PubMed2006

Ultra-fast mass fingerprinting by high-affinity capture of peptides and proteins on derivatized poly(glycidyl methacrylate/divinylbenzene) for the analysis of serum and cell lysates.

The development of support materials in mass fingerprinting is an important task required for diagnostic markers in conjunction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The material-based approach, which we introduced as material-enhanced laser desorption/ionization (MELDI), focuses not only on different functionalities, but also emphasizes the morphology, i.e. porosity or particle size of the carrier material. As a result, it provides a quick and sensitive platform for effective binding of peptides and proteins out of different biofluids, e.g. serum, spinal fluid, urine or cell lysates, and to subsequently analyze them with MALDI-TOF MS. This approach includes a built-in desalting step for serum protein profiling and is sensitive enough to detect proteins and peptides down to 100 fmol/microL. Here we co-polymerized glycidyl methacrylate (GMA) with divinylbenzene (DVB) using thermal polymerization to yield a GMA/DVB polymer for further modifications. Different affinities have been created, such as immobilized metal ion affinity (IDA-Cu2+), reversed-phase (RP) and anion-exchanger (AX) chromatography. The diverse derivatizations and the dispersity of the particles created by different chemical synthetic approaches were confirmed by characteristic infrared (IR) peaks. The polymerization carried out by non-stirring yielded an average pore radius of 6.1 microm (macro-pores) that enhanced the binding capacity enormously by offering enlarged surface areas. Moreover, atomic absorption spectrometry (AAS) provided the metal content loaded on iminodiacetic acid (IDA) in the case of poly(GMA/DVB)-IDA-Cu2+. To summarize, the optimized MELDI approach is sensitive in its performance, extremely fast and can be adapted to robotic systems for routine analysis, allowing sample preparation in less than 5 min in contrast to the conventional surface-enhanced laser desorption/ionization (SELDI) methods.

Blood Proteins↗

PubMed2005

Quantitative detection of phosphoproteins by combination of two-dimensional difference gel electrophoresis and phosphospecific fluorescent staining.

Here we combine a standard two-dimensional difference gel electrophoresis (DIGE) protocol with subsequent post-staining of gels with phosphospecific fluorescent Pro-Q Diamond dye. The combination of these two methods for fluorescence detection of proteins allows quantitative detection of phosphoproteins in 2-DE-gels. We established this protocol within a functional proteomics experiment. Mammary epithelial cells (EpH4) were stimulated in culture by epidermal growth factor (EGF), endosomal fractions prepared after subcellular fractionation and phosphorylated proteins successfully detected on endosomes. For instance, Endo A cytokeratin, known as phosphoprotein and differentiation marker inducible by MAPK signaling, was identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). With this protocol, all steps of combined proteome and phosphoproteome profiling experiments are significantly simplified and accelerated, taking full advantage of both methods in terms of specificity, sensitivity and accuracy of quantification.

Animals↗

PubMed2005

Enrichment of low-abundant serum proteins by albumin/immunoglobulin G immunoaffinity depletion under partly denaturing conditions.

We present a simple protocol for affinity depletion to remove the two most abundant serum proteins, albumin and immunoglobulin G (IgG). Under native conditions, albumin/IgG were efficiently removed and several proteins were enriched as shown by two-dimensional electrophoresis (2-DE). Besides that, partly denaturing conditions were established by adding 5 or 20% acetonitrile (ACN) in order to disrupt the binding of low-molecular-weight (LMW) proteins to the carrier proteins albumin/IgG. 2-DE results showed that the total number of detected LMW proteins increased under denaturing conditions when compared to native conditions. Interestingly, the presence of 5% ACN in serum revealed better enrichment of LMW proteins when compared to 20% ACN condition. Seven randomly distributed spots in albumin/IgG depleted serum samples under 5% ACN condition were picked from the 2-DE gels and identified by mass spectrometry (MS). The intensity of five LMW protein spots increased under denaturing conditions when compared to native conditions. Three of the seven identified spots (serum amyloid P, vitamin D-binding protein, and transthyretin) belong to a group of relatively low-abundant proteins, which make up only 1% of all serum proteins. The method presented here improves the resolution of the serum proteome by increasing the number of visualized spots on 2-D gels and allowing the detection and MS identification of LMW proteins and proteins of lower abundance.

Acetonitriles↗

PubMed2005

Phosphoproteomic analysis using immobilized metal ion affinity chromatography on the basis of cellulose powder.

Detailed characterization of phosphoproteins as well as other post-translationally modified proteins such as glycoproteins, is required to fully understand protein function and regulatory events in cells and organisms. Therefore, an experimental strategy for the isolation of phosphoproteins using a new immobilized metal ion affinity chromatograph (IMAC) material on the basis of cellulose has been developed and characterized. Different approaches have been used to test the material. Recovery rates were determined by 32P labelling of a myelin basic protein fragment and by reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry using a tryptic digest of the model protein bovine beta-casein. Selectivity was demonstrated by enrichment and separation of phosphopeptides from different samples, such as from a digest of horse myoglobin as well as from a digest of in vitro phosphorylated extracellular signal regulates kinase 2 (ERK2) mixed with synthetic phosphopeptides, phosphorylated on different amino acid residues. Furthermore, simplification and optimization of sample pretreatment was achieved by combining the separating (IMAC) and desalting (C18) step during preparative high performance liquid chromatography. The comparison between our material and a commercially available IMAC system (POROS 20 MC; Perspective BioSystems) emphasizes the competitiveness of the cellulose. Confirmed by the obtained data, the cellulose material performed as well as the commercially available sorbent, however with the advantage, that it can be produced rather easily and at very low cost.

Animals↗

PubMedDate not supplied

Poly(glycidyl methacrylate/divinylbenzene)-IDA-FeIII in phosphoproteomics.

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).

Amino Acid Sequence↗

PubMedDate not supplied

Derivatized cellulose combined with MALDI-TOF MS: a new tool for serum protein profiling.

Providing a rapid and sensitive protein profiling method for biomarker discovery from a variety of biological samples is crucial for the introduction of new markers that improve cancer patient diagnosis at early tumor stages, thus increasing the chances of curative treatment. We report here the development and application of derivatized cellulose particles for selective serum protein profiling. For immobilized metal ion affinity chromatography (IMAC), cellulose was derivatized with glycidyl methacrylate (GMA) and subsequently with iminodiacetic acid (IDA). To investigate the application of this material for generating protein profiles of human serum samples, the serum samples were agitated with the derivatized cellulose particles to a suspension and incubated for 2 h at 30 degrees C. After washing, 1 microL of the IDA-Cu(2+)-cellulose suspension was applied directly onto a MALDI-target, mixed with sinapinic acid (SA) and analyzed with MALDI-TOF MS. Consistent serum specific data were obtained from aliquoted samples analyzed several times, indicating the reliability of the method. However, the serum fingerprints obtained proved to be specific for any given serum. The technique presented allows a high enrichment of sample on the developed target leading to a high sensitivity and reproducibility without depletion of albumin and immunoglobulin, and sample elution prior to MS-analysis. The study demonstrates for the first time that derivatized cellulose particles combined with MALDI-TOF MS represent a simple, economical, and rapid approach to generate serum protein profiles for biomarker identification.

Biomarkers↗