Surface-enhanced laser desorption-ionization retentate chromatography mass spectrometry (SELDI-RC-MS): a new method for rapid development of process chromatography conditions.

Protein biochip arrays carrying functional groups typical of those employed for chromatographic sorbents have been developed. When components of a protein mixture are deposited upon an array's functionalized surface, an interaction occurs between the array's surface and solubilized proteins, resulting in adsorption of certain species. The application of gradient wash conditions to the surface of these arrays produces a step-wise elution of retained compounds akin to that accomplished while utilizing columns for liquid chromatography (LC) separations. In retentate chromatography-mass spectrometry (RC-MS), the "retentate" components that remain following a wash are desorbed and ionized when a nitrogen laser is fired at discrete spots on the array after treatment with a laser energy-absorbing matrix solution. Ionized components are analyzed using a time-of-flight mass spectrometer (TOF MS). The present study demonstrates that protein biochips can be used to identify conditions of pH and ionic strength that support selective retention-elution of target proteins and impurity components from ion-exchange surfaces. Such conditions give corresponding behavior when using process-compatible chromatographic sorbents under elution chromatography conditions. The RC-MS principle was applied to the separation of an Fab antibody fragment expressed in Escherichia coli as well as to the separation of recombinant endostatin as expressed in supernatant of Pichia pastoris cultures. Determined optimal array binding and elution conditions in terms of ionic strength and pH were directly applied to regular chromatographic columns in step-wise elution mode. Analysis of collected LC fractions showed favorable correlation to results predicted by the RC-MS method.

Protein biochips for differential profiling.

Progress has been made in utilizing ProteinChip technology to profile and compare protein expression in normal and diseased states, particularly in the areas of cancer, infectious disease and toxicology. The past year has also seen the development of several novel chip types designed to analyze proteins in a fashion analogous to the array-based format of DNA microarrays. Some of these platforms may be used for differential profiling.

Limited acid hydrolysis as a means of fragmenting proteins isolated upon ProteinChip array surfaces.

ProteinChip array technology enables protein purification, protein profiling, and biomarker discovery on a convenient biochip platform. Traditional proteomic approaches towards protein identification rely upon the generation of peptides through the use of specific proteases. However, for a variety of reasons, the digestion of proteins bound to planar arrays by specific proteases, such as trypsin, has proven to be difficult, at times providing little or no protein digestion at all. Additionally, should more than one protein be present on the array surface, the digestion product consists of peptides from different proteins, adding another dimension of complexity to database mining approaches. These factors have driven our group to explore alternative means of on-chip protein digestion. In this article, we describe an approach to generate peptide maps by limited acid hydrolysis. Depending upon the adsorbed protein, this method requires between 500 femtomole to 5 picomole of protein for on-chip hydrolysis. Besides generating several internal peptide fragments, limited acid hydrolysis also has the advantage of generating peptide ladders from the N- or C-terminus of the protein. From these ladders, partial primary sequence of the protein can be directly derived when analyzed by a simple laser desorption/ionization mass spectrometer. Furthermore, tandem mass spectrometry can be performed on several internal peptide fragments, thus facilitating the identification of several proteins within a mixture. Based upon the preliminary results of this work, we continue to explore the possibility of using limited acid hydrolysis to identify unknown proteins captured on ProteinChip array surfaces.

Recent advancements in surface-enhanced laser desorption/ionization-time of flight-mass spectrometry.

The overall history and recent advances in surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) technology is reviewed herein. Fundamentals of SELDI-TOF analysis are presented while drawing comparisons with other laser-based mass spectrometry techniques. The application of SELDI-TOF-MS to functional genomics and biomarker discovery is discussed and exemplified by elucidating a biomarker candidate for prostatic carcinoma. Finally, a short discussion regarding future SELDI requirements and developments is supplied.

Recent trends in protein biochip technology.

This article presents current trends and advances in protein biochip technologies that rely upon extraction and retention of target proteins from liquid media. Analytical strengths as well as technical challenges for these evolving platforms are presented with particular emphasis on selectivity, sensitivity, throughput and utility in the post-genome era. A general review of protein biochip technology is provided, which delineates approaches for protein biochip format and operation, as well as protein detection. A focused discussion of three protein biochip technologies, Biomolecular Interaction Analysis (Biacore, Uppsala, Sweden), Surface Enhanced Laser Desorption/Ionisation (SELDI) ProteinChip Arrays (Ciphergen Biosystems, Fremont, CA, USA) and Fluorescent Planar Wave Guide (Zeptosens, Witterswil, Switzerland), follows along with examples of relevant applications.

Functional wave time-lag focusing matrix-assisted laser desorption/ionization in a linear time-of-flight mass spectrometer: improved mass accuracy.

A strength of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is its ability to analyze mixtures without separation. MALDI mass spectrometers capable of providing a linear mass calibration over a broad mass range should find wide use in these applications. This work addresses issues pertinent to mass measurement accuracy of a time-lag focusing MALDI time-of-flight instrument and presents a new approach to improving mass accuracy by using a functional wave extraction pulse, instead of a square wave, for time-lag focusing. A model is described of an ideal extraction pulse shape that provides constant total kinetic energy for all ions. If total kinetic energy is constant, then there is an exact linear correlation between ion mass and flight time raised to the second power. Using a descending wave extraction pulse, it is demonstrated that mass accuracy of better than 30 ppm using two internal calibrants and better than 70 ppm using external calibrants can be obtained over a 25 ku mass range. The practical aspects of an instrument needed to obtain consistent mass accuracy is discussed. It is found that ion flight time shows a small dependence upon laser flux; flight times increase slightly as the flux increases. But this dependence is much smaller than is observed in continuous-extraction MALDI.

Accurate mass measurement of oligonucleotides using a time-lag focusing matrix-assisted laser desorption/ionization time-of-flight mass spectrometer.

A method for accurate mass measurement of oligonucleotides up to a DNA 35-mer based on matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is described. In this method, a time-lag focusing time-of-flight mass spectrometer is used to achieve high mass resolution to resolve adduct ions that often complicate the mass analysis of oligonucleotides. Mass resolutions between 1170 and 1300 (full width at half maximum) for a 17-mer, 23-mer, and 35-mer are obtained using a 1 m linear time-of-flight instrument with a total sample loading of less than 10 pmol. The effects of sample preparation, type of calibrant and matrix used on the accuracy of mass measurement, based on external calibration, are discussed. A sample preparation protocol that forms a thin film of matrix and sample crystals on a MALDI probe is described. It is shown that mass measurement error less than 100 ppm with reproducibility better than +/-60 ppm can be obtained with either proteins or DNA fragments as external calibrants. Accurate mass measurement for a mixture of DNA fragments is also illustrated.