Protein Chips with SELDI

Because of the washing step, disruptive ions or detergents are absent. More stiU the proteins are adsorbed on the surface linearly and in one layer. Hence, SELDI produces uniformly distributed and imiformly oriented crystals. No unordered crystal salad anymore, as with the other MALDI techniques. The uniform crystal coating produces an excellent and fairly reproducible signal the peak heights vary between successive runs of the identical sample only between 10 and 30%. 

With chips not coated covalently with proteins, you can wash matrix and sample residues off after measuring and then reuse the chip. However, for important and clean experiments you should use new chips. Which new possibilities does SELDI offer  

You can investigate protein-protein interactions (see also Sections 2.5 and 2.2.S.2). Example you have purified or expressed a protein X and you would like to know its function. Neither the sequence nor the purification provides any clues. It would be cool to know which proteins bind to protein X. Maybe you know their function, which would give you valuable pointers. Thus, you couple your protein X to a SELDI chip, if possible under different conditions. Then you wash off the unbound protein X and block unused groups. Now you incubate the chip holes with cell extract or wherever else you suspect binding partners to occur. Wash it off. Co-crystallize with matrix. Off with the chip into the M ALDl-TOF. After a few seconds you know whether a protein binds to protein X. Now this raises the question Is this protein the physiological binding partner To decide this, you need controls. In a control chip hole, you couple a protein that is similar to protein X in isoelectric point and MW. This hole should bind no proteins, or at least no other proteins. On the other hand, cell extracts or serums known not to contain binding partners for protein X should leave no trace in the mass spectrum. Furthermore, the protein should bind to X somewhat stoichiometrically. 

Is everything correct Then you identify the binding partner in databases and order a bottle of champagne. However, if you fail you pay dearly—in the word sense. Failure does not mean that no partner exists. In the cell, every Jack will find his Jill. It is more likely that your conditions were not right, either with the coupling of protein X to the chip or with the incubation of the coated chip with the cell extract. Thus, you have to try out one condition after the other and purchase one chip after the other. Each chip costs 70. 

Another possible application of SELDI comes with the included analysis software. With it you can determine the quantitative differences between two spectra (e.g., ill/healthy or drug/without-drug). This makes sense with SELDI, because the spectra are fairly reproducible 

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The almost endless possibilities of mass spectrometry have been increased by yet another hopeful variant SELDI (surface-enhanced laser desorption ionization). SELDI combines protein chips with a UV-MALDl-TOF. The chips are solid aluminum strips coated with cationic or anionic ion exchangers, with hydrophile or hydrophobic molecules. Chips with activated surfaces are also available. These bind proteins covalently via their amino groups and enable you to coat chips with antibodies or receptors as you need them. Every chip has eight coated holes with a diameter of 1 mm. You apply the sample into the holes. Part of the pro-teins/peptides is adsorbed. The remainder is washed off. The adsorbed proteins/peptides are transferred into matrix and can then be analyzed in the mass spectrometer (Figure 7.8). What can SELDI do better than MALDI ... 

Protein Chip technology, it will be possible to simultaneously analyze protein profiles of body fluids such as serum and urine samples very rapidly. The SELDI mass spectrometry in conjunction with bioinformatics tools could greatly facilitate the discovery of new and improved toxicologic biomarkers. 

Two hundred and forty-eight serum samples provided from the National Ovarian Cancer Early Detection Program clinic at Northwestern University Hospital (Chicago, IlUnios) were analyzed on the same ProteinChip arrays using both a PBS-II and a Qq-TOF MS fitted with a SELDI interface. The proteomic patterns of the serum samples were acquired on the PBS-II TOF MS, immediately followed by their acquisition on the Qq-TOF MS. The key to this study is that the identical set of serum samples was analyzed on the exact same protein-chip surface, eliminating all experimental variability other than the use of two different instruments. 

Surface enhanced laser desorption ionisation (SELDI) is a version of MALDI with the addition of a special chip from which sample components are ionised and desorbed. The chip can be a protein chip array composed of spots of different chromatographic surfaces (which are chemical or biochemical in nature) designed to retain specific proteins. Its main advantage is inclusion of this separation/selection step for crude or complex samples prior to MS analysis. 

In 1993, Hutchens and co-workers described surface-enhanced laser desorption/ionization (SELDI) technique, an affinity technology, which has progressed over the last decade to become a powerful analytical, an on-plate approach (Hutchens and Yip 1993). SELDI is a distinctive form of laser desorption/ionization (LDI) mass spectrometry in which the EDI probe plays an active role in the homogenization, preconcentration, amplification, purification, extraction, enrichment digestion, derivatization, synthesis, separation, and detection with complementary techniques, prior to the desorption and ionization of the analytes by MALDI (Merchant and Weinberger 2000). The principle of this approach is very simple. Biomolecules are captured by adsorption, partition, electrostatic interaction, or affinity chromatography on a solid-phase protein chip surface. Although SELDI provides a unique sample preparation platform, it is similar to MALDI-MS in that a laser... 

The ProteinChip System from Ciphergen Biosystems uses patented SELDI (Surface-Enhanced Laser Desorption/Ionization) ProteinChip technology to rapidly perform the separation, detection, and analysis of proteins at the femtomole level directly from biological samples. ProteinChip Systems use ProteinChip Arrays which contain chemically (cationic, anionic, hydrophobic, hydrophilic, etc.) or biochemically (antibody, receptor, DNA, etc.) treated surfaces for specific interaction with proteins of interest. Selected washes create on-chip, high-resolution protein maps. This protein mass profile, or reten-tate map of the proteins bound to each of the ProteinChip Array surfaces, is quantitatively detected in minutes by the ProteinChip Reader. 

Matrix-assisted laser desorption ionization (MALDI) and surface-enhanced laser desorption ionization (SELDI) have been used online with TOF-MS for protein differential profiles of intact or hydrolyzed biological matrices in proteomics. The potential use of affinity chips, grafted with specific Ab towards the drug compound for MALDI or SELDI, will bring sensitive and selective tools for macromolecules. Specific Ab towards either the intact protein or several signature peptides... 

Figure 6 The SELDI technology. This type of proteomic analytical tool is a class of mass spectroscopy instrument that is useful in high-throughput proteomic fingerprinting of serum. Using a robotic sample dispenser, 1 p,L of serum is applied to the surface of a protein-binding chip. A subset of the proteins in the sample binds to the surface of the chip. The bound proteins are treated with a matrix-assisted laser desorption and ionization matrix and are washed and dried. The chip, which contains multiple patient samples, is inserted into a vacuum chamber where it is irradiated with a laser. The laser desorbs the adherent proteins and causes them to be launched as ions. The TOF of the ion before detection by an electrode is a measure of the mass-to-charge (m/z) value of the ion. The ion spectra can be analyzed by computer-assisted tools that classify a subset of the spectra by characteristic patterns of relative intensity (adapted from www.evmsdoctors.com).

A comparative study of malignant and normal endometrial tissues yielded a panel of proteins displaying differential expression in malignant tissues. A prominent putative marker was identified as chaperonin 10 by both MALDI-Qq-TOF and ESI-Qq-TOF-MS, confirmed by Western blot and immunohistochemistry [73], A comparison of sera of patients with endometrial cancer with those of healthy females using SELDI-TOF-MS (weak cation exchange chips) yielded a number of putative biomarkers upon evaluations with three data mining tools (a tree classifier, Biomarker Wizard, and Biomarker Patterns System). The diagnostic pattern combined with 13 putative markers made it possible to differentiate patients with endometrial cancer from healthy subjects with specificity of 100% and sensitivity of 92.5% [74]. 

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