ProteinChip arrays

Davies, H., Lomas, L., and Austen, B. (1999). Profiling of amyloid b peptide variants using SELDI ProteinChip arrays. BioTechniques 27, 1258-1261,... 

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. 

Diamond DL, Zhang Y, Gaiger A, et al. Use of ProteinChip array surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) to identify thymosin beta-4, a differentially secreted protein from lymphoblastoid cell lines. J. Am. Soc. Mass Spectrom. 2003 14 760-765. 

Austen BM, Frears ER, Davies H. The use of solid ProteinChip arrays to monitor production of Alzheimer s beta amyloid in transfected cells. J Pept Sci 2000 6 459-469. 

Cordingley HC, Roberts SL, Tooke P, Armitage JR, Lane PW, Wu W, Wildsmith SE. Multifactorial screening design and analysis of SELDI-TOF ProteinChip array optimization experiments. Biotechniques 2003 34 364-365, 368-373. 

ProteinChip arrays with preactivated surfaces are also available for covalently coupling of a specific bait molecule (protein, DNA, RNA). This allows the investigation of specific protein interactions such as DNA-protein, receptor-ligand, and antibody-antigen (Ab-Ag), the latter of which permits the generation of a standard curve and hence quantitation studies (21). 

Capture and Quantitation of Aft Using Antibody-Coated ProteinChip Arrays... 

Load 2 il, of anti-A(3 Ab or control Ab (0.1-0.5 mg/mL) to each spot of a preactivated ProteinChip array. As PS 10 and PS20 arrays are commonly used for A(3 capture experiments, analysis of these chip types will be described. 

Fig. 2. Capture of A(3 using Ab-coated ProteinChip arrays. Preactivated arrays with exposed active sites are shown in (A). Ab are covalently bound to the array via amine groups (B). Exposed active sites are blocked using Tris-containing buffer (C). Samples are added to the array, incubated for 1 1 h (D), and washed to remove nonspecifically bound proteins (E). EAM is applied to each spot (F), and the array is introduced into the ProteinChip reader where the laser is fired onto the chip surface. Proteins retained on the array surface are subsequently resolved via TOF-MS, which displays the mass-to-charge value and signal intensities of each protein (G, H). (Adapted from ProteinChip technology training course, Bio-Rad Laboratories.)...

Bradbury, L.E., LeBlanc, J.F. and McCarthy, D.B. (2004) ProteinChip array-based amyloid beta assays. Methods Mol. Biol. 264, 245-257. 

Claussen, U., Eggeling, F. (2005). Identification of protein pattern in kidney cancer using ProteinChip arrays and bioinformatics. Int.J. Mol. Med. 15, 285-290. 

The use of SELDI ProteinChip array technology in 29. renal disease research. Methods Mol. Med. 2003, 86,... 

The most popular MS instrument to perform SELDI-TOF MS analysis is the PBS-II, manufactured by Ciphergen Biosystems Inc. [16], The SELDI-TOF MS instrument is composed of three major components the ProteinChip arrays, the mass analyzer, and the data-analysis software. 

FIGURE 4.3 The variety of ProteinChip arrays available for sample preparation. (A) The upper arrays represent chemically modified chromatographic surfaces, while the bottom arrays are biochemically modified surfaces. Chemically modified surfaces are used to retain a group of proteins, while biochemically modified surfaces are typically used to isolate a specific protein or functional class of proteins. (B) Protein profile of a cell lysate on different ProteinChip surfaces. As shown in the figure for a selection of protein chips, the individual surfaces retain different groups of proteins, depending on their physiochemical properties. The proteins retained are also dependent on the pH of the sample for the cation and anion exchange surfaces. 

FIGURE 4.4 Schematic diagram of the SELDI Ciphergen mass spectrometer. After sample preparation, the ProteinChip arrays are analyzed by a laser-desorption/ionization (LDI) time-of-flight mass spectrometer (TOF MS). The TOF MS measures the molecular weights of the various proteins that are retained on the array. For comparison purposes, the software associated with the SELDI Ciphergen instrument is capable of displaying the resultant data as either a spectral, map, or gel view. 

FIGURE 4.5 Representative spectrum examples of SELDI analysis of pancreatic juice samples bound to IMAC-3 cupper ProteinChip array. A peak of 16,570 Da (arrow) was present in the four pancreatic juice samples from patients with pancreatic adenocarcinoma (PC4, PCS, PC 18, PC24) but absent in the four patients with other pancreatic diseases (IPMN islet cell tumor (ICT) serous cystadenoma (SC)). (Reprinted from Rosty et al. [26], used with permission from the American Association of Cancer Research.)... 

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. 

Fig. 8.1 The different types of ProteinChip Arrays. The spots on the chromatographic ProteinChip Arrays possess hydrophobic, cationic, anionic, metal ion affinity, or hydrophilic properties. These chemical surfaces are best suited for protein expression profiling studies. Another series of Pro-...

The basic procedure for Expression Difference Mapping studies with the Pro-teinChip System is quite simple. Virtually any type of protein-containing solution can be applied to the spots of the ProteinChip Arrays. These spots present either chromatographic surfaces with certain physicochemical characteristics (hydrophobic, cationic, anionic, metal ion-presenting or hy-... 

Typically, for Expression Difference Mapping experiments, chromatographic surfaces are used. The sample requirement is low (1-10 pg total protein per spot), and the sample volume can be freely chosen from 0.5 pL up to around 300 pL. After a short incubation period, unbound proteins and any contaminants on the spot surface are washed away. A solution of energy-absorbing molecules is applied to each of the spots, and the ProteinChip Array is ready for the analysis in the ProteinChip Reader, a highly sensitive laser desorption/ioniza-tion TOF-MS instrament. The results are initially visualized in a graph, with the mass... 

Fig. 8.2 Sample analysis in the ProteinChip Reader. After loading the ProteinChip Array into the ProteinChip Reader, a laser beam is directed onto the sample on the spot. Thereby protons are transferred onto the peptides and proteins that are subsequently accelerated by electromagnetic fields through a flight tube. The time-of-flight is...

ProteinChip Arrays are available with different chromatographic properties, inciuding hydrophobic, hydrophiiic, anion exchange, cation exchange, and immobilized-nnetai affinity surfaces. Other Protein Chip Arrays with p re-activated surfaces are available for covalently coupling protein, DNA, RNA or other bait" molecules by the user. 

Crude biological samples can be applied directly to the ProteinChip Arrays. Application can be done manually by pipetting or by employing Ciphergen s customized configuration of the Beckman Coulter Biomek 2000 laboratory automation station. 

After a short incubation period, unbound proteins are washed off the surface of the ProteinChip Array. Oniy proteins interacting with the chemistry of the array surface are retained for analysis. After washing, energy absorbing molecules are applied to the array as a final step. 

ProteinChip Arrays are then analyzed in the ProteinChip Reader, a time-of-flight mass spectrometer. The mass values and signal intensities for the detected proteins and peptides can be viewed in several formats and then transferred to Ciphergen s software suites for further in-depth analysis. 

The ProteinChip System Series 4000 Enterprise AutoBiomarker Edition, comprised a ProteinChip Reader with autoloader capacity to automatically run - simultaneously - up to 168 bar-code-labeled ProteinChip Arrays with, in total, 1344 samples, a customized Biomek 2000 hquid handling robot and CiphergenExpress Data Manager Software for automated sample tracking and advanced data analysis together with the Biomarker Patterns Software for the direct develop-... 

Fig. 8.4 Hardware components of the ProteinChip System. The ProteinChip Arrays (1) are arranged in bioprocessors to match the 96-well microtiter plate format (2). Samples can be applied using a customized Biomek 2000 liquid-handling robot...

The ProteinChip System Series 4000 Enterprise Edition (4) automatically runs up to 168 barcode labeled ProteinChip Arrays with, in sum,... 

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