Chip-based nanoelectrospray mass spectrometry for protein characterization

In the last several years, significant progress has been made in the development of microfluidic-based analytical technologies for proteomic and drug discovery applications. Chip-based nanoelectrospray coupled to a mass spectrometer detector is one of the recently developed analytical microscale technologies. This technology offers unique advantages for automated nanoelectrospray including reduced sample consumption, improved detection sensitivity and enhanced data quality for proteomic studies. This review presents an overview and introduction of recent developments in chip devices coupled to electrospray mass spectrometers including the development of the automated nanoelectrospray ionization chip device for protein characterization. Applications using automated chip-based nanoelectrospray ionization technology in proteomic and bioanalytical studies are also extensively reviewed in the fields of high-throughput protein identification, protein post-translational modification studies, top-down proteomics, biomarker screening by pattern recognition, noncovalent protein–ligand binding for drug discovery and lipid analysis. Additionally, future trends in chip-based nanoelectrospray technology are discussed.     

Characterization of protein glycosylation using chip-based infusion nanoelectrospray linear ion trap tandem mass spectrometry.

Mass spectrometry (MS) has the potential to revolutionize structural glycobiology and help in the understanding of how post-translation events such as glycosylation affect protein activities. Several approaches to determine the structure of glycopeptides have been used successfully including fast atom bombardment, matrix-assisted laser desorption ionization, and electrospray ionization with a wide variety of mass analyzers. However, the identification of glycopeptides in a complex mixture still remains a challenge. The source of this challenge is primarily due to the poor ionization efficiency and rapid degradation of glycopeptides. In this report we describe the use of a chip-based infusion nanoelectrospray ionization technique in combination with a recently developed linear ion trap for identification and characterization of glycosylation in complex mixtures. Two standard synthetic glycans were analyzed using multiple-stage fragmentation analysis in both positive and negative ionization modes. In addition, the high mannose type N-glycosylation in ribonuclease B (RNase B) was used to map the glycosylation site and obtain the glycan structures. We were able to map the glycosylation site and obtain the glycan structures in RNase B in a single analysis. The results reported here demonstrate that the fully automated chip-based nanoelectrospray linear ion trap platform is a valuable system for oligosaccharide analyses due to the unique MS/MS and MS(n) capability of the linear ion trap and the extended analysis time provided by the ionization technique.     

Rapid parallel mutation scanning of gene fragments using a microelectronic protein-DNA chip format

We have developed a method for the de novo discovery of genetic variations, including single nucleotide polymorphisms and mutations, on microelectronic chip devices. The method combines the features of electronically controlled DNA hybridisation on open-format microarrays, with mutation detection by a fluorescence-labelled mismatch- binding protein. Electronic addressing of DNA strands to distinct test sites of the chip allows parallel analysis of several individuals, as demonstrated for mutations in different exons of the p53 gene. This microelectronic chip-based mutation discovery assay may substitute for time-consuming sequencing studies and will complement existing technologies in genomic research.     

Rapid parallel mutation scanning of gene fragments using a microelectronic protein–DNA chip format

We have developed a method for the de novo discovery of genetic variations, including single nucleotide polymorphisms and mutations, on microelectronic chip devices. The method combines the features of electronically controlled DNA hybridisation on open-format microarrays, with mutation detection by a fluorescence-labelled mismatch- binding protein. Electronic addressing of DNA strands to distinct test sites of the chip allows parallel analysis of several individuals, as demonstrated for mutations in different exons of the p53 gene. This microelectronic chip-based mutation discovery assay may substitute for time-consuming sequencing studies and will complement existing technologies in genomic research.     

Characterization of protein glycosylation using chip-based nanoelectrospray with precursor ion scanning quadrupole linear ion trap mass spectrometry.

Glycosylation is one of the most important posttranslational modifications affecting the functions of proteins and cell activities. Mass spectrometry (MS) has proven to be an effective tool for structural glycobiology and has helped gain an understanding of glycoprotein-mediated diseases. Although electro-spray ionization-tandem MS remains widely recognized as an effective means for oligosaccharide characterization, the hydrophilic nature of glycans has often caused the poor ionization efficiency requiring either derivatization or nanoelectrospray to improve detection sensitivity. In this report we describe the use of a chip-based infusion nanoelectrospray platform coupled with the hybrid triple quadrupole/linear ion trap for identification and characterization of glycosylation in complex mixtures. The high-mannose-type N-glycosylation in ribonuclease B was used to map the glycosylation site and obtain glycan structures. Using the chip-based nanoelectro-spray with precursor ion scanning linear ion trap MS, we were able to map the glycosylation site and obtain the glycan structures in ribonuclease B at 100 fmol/microL in a single analysis. In addition, a new, low-abundant glycoform with an additional hexose (Hex10GlcNAc2) attached to ribonuclease B was discovered. The results reported here demonstrate that the chip-based infusion nanoelectrospray ionization coupled to a quadrupole/linear ion trap platform is a valuable system, as it provides high sensitivity and stability for nanoelectrospray analysis, and allows extended acquisition time for completing precursor ion scanning and subsequent MS2 and MS3 information in a single analysis.     

2D protein electrophoresis: can it be perfected?

23 years after O'Farrel developed two-dimensional gel electrophoresis we still debate if the technique can be improved, or if there are other alternative separation technologies that can challenge its central position in proteomic projects. These questions are relevant as the pharmaceutical industry expects proteomic studies to provide novel protein targets for drug discovery and diagnostics. In our opinion, there are various aspects of the technology that can be improved, including resolution, sample preparation and detection, but so far there is no alternative technique(s) available, or any under development, that can replace it.     

Poxvirus Proteomics and Virus-Host Protein Interactions

Summary: Studies of the functional proteins encoded by the poxvirus genome provide information about the composition of the virus as well as individual virus-virus protein and virus-host protein interactions, which provides insight into viral pathogenesis and drug discovery. Widely used proteomic techniques to identify and characterize specific protein-protein interactions include yeast two-hybrid studies and coimmunoprecipitations. Recently, various mass spectrometry techniques have been employed to identify viral protein components of larger complexes. These methods, combined with structural studies, can provide new information about the putative functions of viral proteins as well as insights into virus-host interaction dynamics. For viral proteins of unknown function, identification of either viral or host binding partners provides clues about their putative function. In this review, we discuss poxvirus proteomics, including the use of proteomic methodologies to identify viral components and virus-host protein interactions. High-throughput global protein expression studies using protein chip technology as well as new methods for validating putative protein-protein interactions are also discussed.     

Lectin capture strategies combined with mass spectrometry for the discovery of serum glycoprotein biomarkers

The application of mass spectrometry to identify disease biomarkers in clinical fluids like serum using high throughput protein expression profiling continues to evolve as technology development, clinical study design, and bioinformatics improve. Previous protein expression profiling studies have offered needed insight into issues of technical reproducibility, instrument calibration, sample preparation, study design, and supervised bioinformatic data analysis. In this overview, new strategies to increase the utility of protein expression profiling for clinical biomarker assay development are discussed with an emphasis on utilizing differential lectin-based glycoprotein capture and targeted immunoassays. The carbohydrate binding specificities of different lectins offer a biological affinity approach that complements existing mass spectrometer capabilities and retains automated throughput options. Specific examples using serum samples from prostate cancer and hepatocellular carcinoma subjects are provided along with suggested experimental strategies for integration of lectin-based methods into clinical fluid expression profiling strategies. Our example workflow incorporates the necessity of early validation in biomarker discovery using an immunoaffinity-based targeted analytical approach that integrates well with upstream discovery technologies.     

Targeted mass spectrometry approaches for protein biomarker verification

The search for protein biomarkers has been a highly pursued topic in the proteomics community in the last decade. This relentless search is due to the constant need for validated biomarkers that could facilitate disease risk stratification, disease diagnosis, prognosis, monitoring as well as drug development, which ultimately would improve our quality of life. The recent development of proteomic technologies including the advancement of mass spectrometers with high sensitivity and speed has greatly advanced the discovery of potential biomarkers. One of the bottlenecks lies in the development of well-established verification assays to screen the biomarker candidates identified in the discovery stage. Recently, absolute quantitation using multiple-reaction monitoring mass spectrometry (MRM-MS) in combination with isotope-labeled internal standards has been extensively investigated as a tool for high-throughput protein biomarker verification. In this review, we describe and discuss recent developments and applications of MRM-MS methods for biomarker verification.     

Biomarker discovery: proteome fractionation and separation in biological samples.

Proteomics, analogous with genomics, is the analysis of the protein complement present in a cell, organ, or organism at any given time. While the genome provides information about the theoretical status of the cellular proteins, the proteome describes the actual content, which ultimately determines the phenotype. The broad application of proteomic technologies in basic science and clinical medicine has the potential to accelerate our understanding of the molecular mechanisms underlying disease and may facilitate the discovery of new drug targets and diagnostic disease markers. Proteomics is a rapidly developing and changing scientific discipline, and the last 5 yr have seen major advances in the underlying techniques as well as expansion into new applications. Core technologies for the separation of proteins and/or peptides are one- and two-dimensional gel electrophoresis and one- and two-dimensional liquid chromatography, and these are coupled almost exclusively with mass spectrometry. Proteomic studies have shown that the most effective analysis of even simple biological samples requires subfractionation and/or enrichment before protein identification by mass spectrometry. Selection of the appropriate technology or combination of technologies to match the biological questions is essential for maximum coverage of the selected subproteome and to ensure both the full interpretation and the downstream utility of the data. In this review, we describe the current technologies for proteome fractionation and separation of biological samples, based on our lab workflow for biomarker discovery and validation.     

Proteomics in biomarker discovery and drug development

Proteomics is a research field aiming to characterize molecular and cellular dynamics in protein expression and function on a global level. The introduction of proteomics has been greatly broadening our view and accelerating our path in various medical researches. The most significant advantage of proteomics is its ability to examine a whole proteome or sub-proteome in a single experiment so that the protein alterations corresponding to a pathological or biochemical condition at a given time can be considered in an integrated way. Proteomic technology has been extensively used to tackle a wide variety of medical subjects including biomarker discovery and drug development. By complement with other new technique advances in genomics and bioinformatics, proteomics has a great potential to make considerable contribution to biomarker identification and to revolutionize drug development process. This article provides a brief overview of the proteomic technologies and their application in biomarker discovery and drug development.     

Modern Tumor Marker Discovery in Urology: Surface Enhanced Laser Desorption and Ionization (SELDI)

During the last two decades, biomarker research has benefited from the introduction of new proteomic analytical techniques. In this article, we review the application of surface enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectroscopy in urologic cancer research. After reviewing the literature from MEDLINE on proteomics and urologic oncology, we found that SELDI-TOF is an emerging proteomic technology in biomarker discovery that allows for rapid and sensitive analysis of complex protein mixtures. SELDI-TOF is a novel proteomic technology that has the potential to contribute further to the understanding and clinical exploitation of new, clinically relevant biomarkers.     

The discovery of stannin in rat dorsal root ganglia using an integrated proteohistological approach

The use of proteomic analysis to discover proteins (previously identified or unknown) in a tissue sample is a valuable tool. However, there is a limit to the extent one can validate a discovery with any single technology. In an effort to obviate this inherent constraint and to add value and dimension to protein profiling, we have coupled the information obtained through proteomic techniques with the validation provided by in situ hybridization and immunohistochemistry techniques. This approach can be illustrated by our efforts in the discovery of stannin in rat dorsal root ganglia (DRG). In this study, we initially used the Ciphergen ProteinChip^® to perform protein profiling on the DRG of rats in a carrageenan-induced paw inflammation study. In an effort to discover new potential targets in inflammatory pain models, we profiled many potential peaks unique to the ipsilateral DRG of interest. One protein, found to bind to a hydrophobic chip at a molecular mass of 9500 Dalton, was preliminarily identified as stannin. To confirm its identification, we performed in situ hybridization and immunohistochemistry on the source DRG tissue to investigate the presence of stannin mRNA and protein expression, respectively. In addition to confirming the presence of stannin in these DRGs, we observed the upregulation of stannin in the DRGs over the course of carrageenan-induced inflammation, suggesting a possible role of stannin in inflammatory hyperalgesia. Taken together, these results illustrate the synergistic benefits of coupling 0 proteomic and histochemical techniques in identifying and validating targets and biomarkers for drug discovery.     

Increased throughput and reduced carryover of mass spectrometry-based proteomics using a high-efficiency nonsplit nanoflow parallel dual-column capillary HPLC system

We report a new design of a fully automated, high-efficiency parallel nonsplit nanoflow capillary HPLC system, coupled on-line with linear ion trap (LTQ) and high performance nanoelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (nanoESI LTQ-FTICR MS). The system, intended for high-throughput proteome analysis of complex protein mixtures, notably serum and plasma, consists of two reversed-phase trap columns for large volume sample injection with high speed sample loading and desalting and two reversed-phase analytical capillary columns. Through a nanoscale two-position, 10-port switching valve, the whole system is terminated by a 10 microm i.d. of nanoemitter mounted on the nanoelectrospray source in front of the sampling cone of the LTQ-FTICR MS. Gradient elution to both nanoflow-rate capillary columns is simultaneously delivered by a single HPLC system via two independent binary gradient pump systems. The parallel capillary column approach eliminates the time delays for column regeneration/equilibration since one capillary column is used for separating the sample mixtures and delivering the separated fractions to the MS, while the other capillary column is being regenerated and equilibrated. The reproducibility of retention time and peak intensity of the present automated parallel nanoflow-rate capillary HPLC system is comparable to that obtained using a single column configuration. Replicate injections of tryptic digests indicated that this system provided good reproducibility of retention time and peak area on both columns with average CV values of less than 1.08% and 7.04%, respectively. Throughput was increased to 100% for 2-h LC-MS analysis compared to the single capillary column LC-MS pipeline. Application of this system is demonstrated in a plasma proteomic study. A total of 312 868 MSMS events were acquired and 1564 proteins identified with high confidence (Protein Prophet > or = 0.9, and peptides matched > or = 2). Comparison of a series of plasma fractions run using the single-column LC-MS versus the parallel-column LC-MS demonstrated that parallel-column LC-MS system significantly reduced the sample carryover, improved MS data quality and increased the number of MS/MS sequence scan events.     

Two-dimensional mass spectrometric mapping

The identification and relative quantification of proteins in closely related biological samples is the backbone for many investigations in systems biology and for the discovery of biomarkers. While two-dimensional gel-based methodologies are still widely used for comparative proteomic studies, the recent advent of gel-free methodologies may allow the analysis of a larger number of samples in an automated fashion. Most of the technologies presented in this review require a chemical modification of proteins before analysis, and rely on the relative intensities of mass spectrometry signals for protein quantification. In particular, two-dimensional mass spectrometric mapping methodologies provide a visual representation of mass spectrometric data, thus facilitating the identification of differences in relative protein abundance.     

Chemical proteomics and its impact on the drug discovery process

Despite the rapid growth of postgenomic data and fast-paced technology advancement, drug discovery is still a lengthy and difficult process. More effective drug design requires a better understanding of the interaction between drug candidates and their targets/off-targets in various situations. The ability of chemical proteomics to integrate a multiplicity of disciplines enables the direct analysis of protein activities on a proteome-wide scale, which has enormous potential to facilitate drug target elucidation and lead drug verification. Over recent years, chemical proteomics has experienced rapid growth and provided a valuable method for drug target identification and inhibitor discovery. This review introduces basic concepts and technologies of different popular chemical proteomic approaches. It also covers the essential features and recent advances of each approach while underscoring their potentials in drug discovery and development.     

Proteomics for cancer drug design

Introduction: Signal transduction cascades drive cellular proliferation, apoptosis, immune, and survival pathways. Proteins have emerged as actionable drug targets because they are often dysregulated in cancer, due to underlying genetic mutations, or dysregulated signaling pathways. Cancer drug development relies on proteomic technologies to identify potential biomarkers, mechanisms-of-action, and to identify protein binding hot spots.

Areas covered: Brief summaries of proteomic technologies for drug discovery include mass spectrometry, reverse phase protein arrays, chemoproteomics, and fragment based screening. Protein-protein interface mapping is presented as a promising method for peptide therapeutic development. The topic of biosimilar therapeutics is presented as an opportunity to apply proteomic technologies to this new class of cancer drug.

Expert opinion: Proteomic technologies are indispensable for drug discovery. A suite of technologies including mass spectrometry, reverse phase protein arrays, and protein-protein interaction mapping provide complimentary information for drug development. These assays have matured into well controlled, robust technologies. Recent regulatory approval of biosimilar therapeutics provides another opportunity to decipher the molecular nuances of their unique mechanisms of action. The ability to identify previously hidden protein hot spots is expanding the gamut of potential drug targets. Proteomic profiling permits lead compound evaluation beyond the one drug, one target paradigm.     

Automated nano-electrospray mass spectrometry for protein-ligand screening by noncovalent interaction applied to human H-FABP and A-FABP

A method for ligand screening by automated nano-electrospray ionization mass spectrometry (nano-ESI/MS) is described. The core of the system consisted of a chip-based platform for automated sample delivery from a 96-well plate and subsequent analysis based on noncovalent interactions. Human fatty acid binding protein, H-FABP (heart) and A-FABP (adipose), with small potential ligands was analyzed. The technique has been compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation with the found hits was obtained. In the current MS screening method, the cycle time per sample was 1.1 min, which is approximately 50 times faster than NMR for single compounds and approximately 5 times faster for compound mixtures. High reproducibility was achieved, and the protein consumption was in the range of 88 to 100 picomoles per sample. Futhermore, a novel protocol for preparation of A-FABP without the natural ligand is presented. The described screening approach is suitable for ligand screening very early in the drug discovery process before conventional high-throughput screens (HTS) are developed and/or used as a secondary screening for ligands identified by HTS.     

Lab-on-a-chip: applications in proteomics

Recent advances in chip-based separation of proteins provide methods that are faster and more convenient than conventional gel electrophoresis. Rapid and automated protein sizing on a chip is at the commercial stage and first attempts have been made to perform two-dimensional separation on a chip. Numerous designs have been described to interface a microfluidic chip to a mass spectrometer. Impressive integration efforts are demonstrated by the ability to perform on-chip trypsin digestion, separation and injection into a mass spectrometer with a single device.     

Advances in shotgun proteomics and the analysis of membrane proteomes

The emergence of shotgun proteomics has facilitated the numerous biological discoveries made by proteomic studies. However, comprehensive proteomic analysis remains challenging and shotgun proteomics is a continually changing field. This review details the recent developments in shotgun proteomics and describes emerging technologies that will influence shotgun proteomics going forward. In addition, proteomic studies of integral membrane proteins remain challenging due to the hydrophobic nature in integral membrane proteins and their general low abundance levels. However, there have been many strategies developed for enriching, isolating and separating membrane proteins for proteomic analysis that have moved this field forward. In summary, while shotgun proteomics is a widely used and mature technology, the continued pace of improvements in mass spectrometry and proteomic technology and methods indicate that future studies will have an even greater impact on biological discovery.