Accelerated identification of proteins by mass spectrometry by employing covalent pre-gel staining with Uniblue A

Background

The identification of proteins by mass spectrometry is a standard method in biopharmaceutical quality control and biochemical research. Prior to identification by mass spectrometry, proteins are usually pre-separated by electrophoresis. However, current protein staining and de-staining protocols are tedious and time consuming, and therefore prolong the sample preparation time for mass spectrometry.

Methodology and Principal Findings

We developed a 1-minute covalent pre-gel staining protocol for proteins, which does not require de-staining before the mass spectrometry analysis. We investigated the electrophoretic properties of derivatized proteins and peptides and studied their behavior in mass spectrometry. Further, we elucidated the preferred reaction of proteins with Uniblue A and demonstrate the integration of the peptide derivatization into typical informatics tools.

Conclusions and Significance

The Uniblue A staining method drastically speeds up the sample preparation for the mass spectrometry based identification of proteins. The application of this chemo-proteomic strategy will be advantageous for routine quality control of proteins and for time-critical tasks in protein analysis.     

Application of Mass Spectrometry in Proteomics

Mass spectrometry has arguably become the core technology in proteomics. The application of mass spectrometry based techniques for the qualitative and quantitative analysis of global proteome samples derived from complex mixtures has had a big impact in the understanding of cellular function. Here, we give a brief introduction to principles of mass spectrometry and instrumentation currently used in proteomics experiments. In addition, recent developments in the application of mass spectrometry in proteomics are summarised. Strategies allowing high-throughput identification of proteins from highly complex mixtures include accurate mass measurement of peptides derived from total proteome digests and multidimensional peptide separations coupled with mass spectrometry. Mass spectrometric analysis of intact proteins permits the characterisation of protein isoforms. Recent developments in stable isotope labelling techniques and chemical tagging allow the mass spectrometry based differential display and quantitation of proteins, and newly established affinity procedures enable the targeted characterisation of post-translationally modified proteins. Finally, advances in mass spectrometric imaging allow the gathering of specific information on the local molecular composition, relative abundance and spatial distribution of peptides and proteins in thin tissue sections.     

Selective Isolation of Glycoproteins and Glycopeptides for MALDI-TOF MS Detection Supported by Magnetic Particles

Glycosylation is the most common form of posttranslational modification of proteins (50–80%). The isolation, discovery, and subsequent identification of glycosylated peptides and proteins is becoming more and more important in glycoproteomics and diagnosis. MALDI-TOF mass spectrometry is an ideal technique for identifying peptides and proteins and their corresponding modifications. The enrichment of glycosylated peptides and proteins from different sources can be attained by affinity chromatography supported by functionalized magnetic particles. Covalent coating of magnetic beads with Concanavalin A (ConA) and diboronic acid was performed by carbodiimide and poly-glutaraldehyde methods, respectively. The functionalized beads were employed to establish and optimize protocols for the binding and detection of glycosylated peptides and proteins with respect to an automated workflow and the subsequent detection and identification by MALDI-TOF mass spectrometry. For several model proteins, the capture and identification could be demonstrated by SDS-PAGE and MALDI-TOF mass spectrometry. According to the type of glycosylation (high man-nose, hybrid, or complex type) the different proteins were enriched by ConA or boronic acid–functionalized beads.     

Improved mass spectrometric identification of gel-separated hydrophobic membrane proteins after sodium dodecyl sulfate removal by ion-pair extraction

Separation and identification of hydrophobic membrane proteins is a major challenge in proteomics. Identification of such sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins by peptide mass fingerprinting (PMF) via matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) is frequently hampered by the insufficient amount of peptides being generated and their low signal intensity. Using the seven helical transmembrane-spanning proton pump bacteriorhodopsin as model protein, we demonstrate here that SDS removal from hydrophobic proteins by ion-pair extraction prior to in-gel tryptic proteolysis leads to a tenfold higher sensitivity in mass spectrometric identification via PMF, with respect to initial protein load on SDS-PAGE. Furthermore, parallel sequencing of the generated peptides by electrospray ionization-mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) was possible without further sample cleanup. We also show identification of other membrane proteins by this protocol, as proof of general applicability.     

Accurate mass tag retention time database for urine proteome analysis by chromatography-mass spectrometry

Information about peptides and proteins in urine can be used to search for biomarkers of early stages of various diseases. The main technology currently used for identification of peptides and proteins is tandem mass spectrometry, in which peptides are identified by mass spectra of their fragmentation products. However, the presence of the fragmentation stage decreases sensitivity of analysis and increases its duration. We have developed a method for identification of human urinary proteins and peptides. This method based on the accurate mass and time tag (AMT) method does not use tandem mass spectrometry. The database of AMT tags containing more than 1381 AMT tags of peptides has been constructed. The software for database filling with AMT tags, normalizing the chromatograms, database application for identification of proteins and peptides, and their quantitative estimation has been developed. The new procedures for peptide identification by tandem mass spectra and the AMT tag database are proposed. The paper also lists novel proteins that have been identified in human urine for the first time.     

Technical aspects of functional proteomics in plants

Since the completion of genome sequences of several organisms, attention has been focused to determine the function and functional network of proteins by proteome analysis. This analysis is achieved by separation and identification of proteins, determination of their function and functional network, and construction of an appropriate database. Many improvements in separation and identification of proteins, such as two-dimensional electrophoresis, nano-liquid chromatography and mass spectrometry, have rapidly been achieved. Some new techniques which include top-down mass spectrometry and tandem affinity purification have emerged. These techniques have provided the possibility of high-throughput analysis of function and functional network of proteins in plants. However, to cope with the huge information emerging from proteome analyses, more sophisticated techniques and software are essential. The development and adaptation of such techniques will ease analyses of protein profiling, identification of post-translational modifications and protein-protein interaction, which are vital for elucidation of the protein functions.     

Improved protein identification through the use of unstained gels

In this work, a method for improved protein identification of low-abundance proteins using unstained gels, in combination with robotics and matrix-assisted laser desorption/ionization tandem mass spectrometry, has been developed and evaluated. Omitting the silver-staining process resulted in increased protein identification scores, an increase in the number of peptides observed in the MALDI mass spectrum, and improved quality of the tandem mass spectrometry data.     

Ion/ion chemistry as a top-down approach for protein analysis

Developing methodology for analyzing complex protein mixtures in a rapid fashion is one of the most challenging problems facing analytical biochemists today. Recent advances in mass spectrometry for the analysis of intact proteins (i.e. the top-down approach) show great promise for rapid protein identification. The ion/ion chemistry approach for the detection and identification of target proteins in complex matrices, determination of fragmentation channels as a function of precursor ion charge state, and post-translational modification characterization are discussed with particular emphasis on tandem mass spectrometry of intact proteins.     

Protein identification using 2D-LC-MS/MS

Multidimensional liquid chromatography techniques have been coupled to tandem mass spectrometry to provide a robust method to identify proteins in complex mixtures. Data acquisition is interfaced directly with search algorithms for identification through cross-correlation with databases. This review describes the most recent advances in methodologies for protein identification by mass spectrometry and describes the limitations of the application of the technologies.     

Enrichment of low-molecular-weight proteins from biofluids for biomarker discovery

The dramatic progress in mass spectrometry-based methods of protein identification has triggered a new quest for disease-associated biomarkers. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and its variant surface-enhanced laser desorption/ionization mass spectrometry, provide effective means to explore the less studied information slice of the human serum proteome -- low-molecular-weight proteins and peptides. These low-molecular-weight proteins and peptides are promising for the detection of important biomarkers. Due to the significant experimental problems imposed by high-abundance and high-molecular-weight proteins, it is important to effectively remove these species prior to mass spectrometry analysis of the low-molecular-weight serum and plasma proteomes. In this review, the advantages afforded by recently introduced methods for prefractionation of serum, as they pertain to the detection and identification of biomarkers, will be discussed.     

Enrichment of low-molecular-weight proteins from biofluids for biomarker discovery

The dramatic progress in mass spectrometry-based methods of protein identification has triggered a new quest for disease-associated biomarkers. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and its variant surface-enhanced laser desorption/ionization mass spectrometry, provide effective means to explore the less studied information slice of the human serum proteome – low-molecular-weight proteins and peptides. These low-molecular-weight proteins and peptides are promising for the detection of important biomarkers. Due to the significant experimental problems imposed by high-abundance and high-molecular-weight proteins, it is important to effectively remove these species prior to mass spectrometry analysis of the low-molecular-weight serum and plasma proteomes. In this review, the advantages afforded by recently introduced methods for prefractionation of serum, as they pertain to the detection and identification of biomarkers, will be discussed.     

Rapid analysis of tryptically digested cerebrospinal fluid using capillary electrophoresis-electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry

Mass spectrometry has in recent years been established as the method of choice for protein identification and characterization in proteomics. Capillary electrophoresis (CE) is a fast and efficient method for the separation of peptides and proteins. The on-line combination of CE with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) has been shown to be a powerful tool in the analysis of complex mixtures of proteins. This paper presents the first results from a proteomic analysis of human cerebrospinal fluid proteins by tryptic digestion and CE-FTICR-MS, where 30 proteins could be identified on a 95% confidence level with mass measurement errors less than 5 ppm.     

Functional genomics by mass spectrometry

Systematic analysis of the function of genes can take place at the oligonucleotide or protein level. The latter has the advantage of being closest to function, since it is proteins that perform most of the reactions necessary for the cell. For most protein based ('proteomic') approaches to gene function, mass spectrometry is the method of choice. Mass spectrometry can now identify proteins with very high sensitivity and medium to high throughput. New instrumentation for the analysis of the proteome has been developed including a MALDI hybrid quadrupole time of flight instrument which combines advantages of the mass finger printing and peptide sequencing methods for protein identification. New approaches include the isotopic labeling of proteins to obtain accurate quantitative data by mass spectrometry, methods to analyze peptides derived from crude protein mixtures and approaches to analyze large numbers of intact proteins by mass spectrometry directly. Examples from this laboratory illustrate biological problem solving by modern mass spectrometric techniques. These include the analysis of the structure and function of the nucleolus and the analysis of signaling complexes.     

Rapid identification of probiotic lactobacillus biosurfactant proteins by ProteinChip tandem mass spectrometry tryptic peptide sequencing

A novel ProteinChip-interfaced tandem mass spectrometer was employed to identify collagen binding proteins from biosurfactant produced by Lactobacillus fermentum RC-14. On-chip tryptic digestion of the captured collagen binding proteins resulted in rapid sequence identification of five novel tryptic peptide sequences via collision-induced dissociation tandem mass spectrometry.     

Evaluation of the compact high-field orbitrap for top-down proteomics of human cells

Mass spectrometry based proteomics generally seeks to identify and fully characterize protein species with high accuracy and throughput. Recent improvements in protein separation have greatly expanded the capacity of top-down proteomics (TDP) to identify a large number of intact proteins. To date, TDP has been most tightly associated with Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. Here, we couple the improved separations to a Fourier-transform instrument based not on ICR but using the Orbitrap Elite mass analyzer. Application of this platform to H1299 human lung cancer cells resulted in the unambiguous identification of 690 unique proteins and over 2000 proteoforms identified from proteins with intact masses <50 kDa. This is an early demonstration of high throughput TDP (>500 identifications) in an Orbitrap mass spectrometer and exemplifies an accessible platform for whole protein mass spectrometry.     

Protein identification by in-gel digestion and mass spectrometric analysis

This protocol details a method for the identification of proteins that have been separated by gel electrophoresis. In-gel digestion of the protein bands with trypsin followed by quadrupole ion-trap or other triple quadrupole mass spectrometry techniques is described. The proteins can be identified by database searching of the mass fingerprint of the intact peptides and of the characteristic fragment masses produced by tandem mass spectrometry.     

Chemical methods for glycoprotein discovery

An important frontier in glycoproteomics is the discovery of proteins with post-translational glycan modifications. The first step in glycoprotein identification is the isolation of glycosylated proteins from the remainder of the proteome. New enzymatic and metabolic methods are being used to chemically tag proteins to enable their isolation. Once isolated, glycoproteins can be identified by mass spectrometry. Additional information can be obtained by using either enzymatic or chemoselective reactions to incorporate isotope labels at specific sites of glycosylation. Isotopic labeling facilitates mass spectrometry-based confirmation of glycoprotein identity, identification of glycosylation sites, and quantification of the extent of modification. By combining chemical tagging for isolation and isotope labeling for mass spectrometry analysis, researchers are developing highly effective strategies for glycoproteomics. These techniques are enabling cancer biologists to identify biomarkers whose glycosylation state correlates with disease states, and developmental biologists to characterize stage-specific changes in glycoprotein expression. Next-generation methods will make functional analyses of the glycoproteome possible, including the discovery of glycoprotein interaction partners and the identification of enzymes responsible for synthesis of particular glycan structures.     

Mass processing--an improved technique for protein identification with mass spectrometry data.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis following tryptic digestion of polyacrylamide gel pieces is a common technique used to identify proteins. This approach is rapid, sensitive, and user friendly, and is becoming widely available to scientists in a variety of biological fields. Here we introduce a simple and effective strategy called "mass processing" where the list of masses generated from a mass spectrometer undergoes two stages of data reduction before identification. Mass processing improves the ability to identify in-gel tryptic-digested proteins by reducing the number of nonsample masses submitted to protein identification database search engines. Our results demonstrate that mass processing improves the statistical score and rank of putative protein identifications, especially with low-quantity samples, thus increasing the ability to confidently identify proteins with mass spectrometry data.     

High-sensitivity analysis of human plasma proteome by immobilized isoelectric focusing fractionation coupled to mass spectrometry identification

Immobilized pH gradients isoelectric focusing (IPG-IEF) is the first dimension typically used in two-dimensional gel electrophoresis (2-DE). It can also be used on its own in conjunction with tandem mass spectrometry (MS/MS) for the analysis of proteins. Here, we described a strategy combining isoelectric focusing in immobilized pH gradient strips, and mass spectrometry to create a new high-throughput and sensitive detection method. Protein mixture is separated by in-gel IEF, then the entire strip is cut into a set of gel sections. Proteins in each gel section are digested with trypsin, and the resulted peptides are subjected to reversed-phase high performance liquid chromatography followed by electrospray-linear ion-trap tandem mass analysis. Using this optimized strategy, we have identified 744 distinct human proteins from an IPG strip loaded only 300 microg of plasma proteins. When compared with other works in published literatures, this study offered a more convenient and sensitive method from gel to mass spectrometry for the separation and identification proteins of complex biological samples.