Automated, on-line two-dimensional nano liquid chromatography tandem mass spectrometry for rapid analysis of complex protein digests

Evaluation of cellular processes and their changes at the level of protein expression and post-translational modifications may allow identification of novel proteins and the mechanisms involved in pathogenic processes. However, the number of proteins and, after tryptic digestion, of peptides from a single cell can be tremendously high. Separation and analysis of such complex peptide mixtures can be performed using multidimensional separation techniques such as two-dimensional gel electrophoresis or two-dimensional-high-performance liquid chromatography (2-D-HPLC). The aim of this work was to establish a fully automated on-line 2-D-HPLC separation method with column switching for the separation of complex tryptic digests. A model mixture of five proteins as well as a nuclear matrix protein sample were digested with trypsin and separated using a strong cation exchange (SCX) column in the first dimension and nano reversed phase in the second dimension. Separated peptides were detected using an ion trap mass spectrometer. The advantages of this new fully automated method are rapid sample loading, the possibility of injecting large volumes and no introduction of salt into the mass spectrometer. Furthermore, column switching allows the independent control and optimization of the two dimensions independently.     

Laser desorption ionization mass spectrometry of protein tryptic digests on nanostructured silicon plates

We report on the simple application of a new nanostructured silicon (NanoSi) substrate as laser desorption/ionization (LDI)-promoting surface for high-throughput identification of protein tryptic digests by a rapid MS profiling and subsequent MS/MS analysis. The NanoSi substrate is easily prepared by chemical etching of crystalline silicon in NH(4)F/HNO(3)/AgNO(3) aqueous solution. To assess the LDI performances in terms of sensitivity, repeatability and robustness, the detection of small synthetic peptides (380-1700Da) was investigated. Moreover, peptide sequencing was tackled. Various tryptic synthetic peptide mixtures were first characterized in MS and MS/MS experiments carried out on a single deposit. Having illustrated the capability to achieve peptide detection and sequencing on these ionizing surfaces in the same run, protein tryptic digests from Cytochrome C, β-Casein, BSA and Fibrinogen were then analyzed in the femtomolar range (from 50 fmol for Cytochrome C down to 2 fmol for Fibrinogen). Comparison of the NanoSi MS and MS/MS data with those obtained with sample conditioned in organic matrix demonstrated a great behavior for low mass responses. We demonstrated the capability of LDI on NanoSi to be a complementary method to MALDI peptide mass fingerprinting ensuring determination of peptide molecular weights and sequences for more efficient protein database searches.     

A gas chromatography–mass spectrometry method to monitor detergents removal from a membrane protein sample

In membrane protein biochemical and structural studies, detergents are used to mimic membrane environment and maintain functional, stable conformation of membrane proteins in the absence of lipid bilayers. However, detergent concentration, esp. molar ratio of membrane protein to detergent is usually unknown. Here, a gas chromatography–mass spectrometry selected ion monitoring (GC–MS-SIM) method was developed to quantify four detergents which are frequently used in membrane protein structural studies. To remove excessive detergents, a filtered centrifugation using Centricon tubes was applied. A membrane protein Ig-Beta fragment in four different detergent micelles was exemplified. Detergent concentrations in the upper and lower fraction of the Centricon tube were measured after each round of centrifugation. The results were very consistent to basic properties of detergent micelles in aqueous solvents. Therefore, coupling of GC–MS-SIM and detergent removal by Centricon tubes, detergents concentration, esp. molar ratio of membrane protein to detergent could be controlled, which will expedite membrane protein structural and biochemical studies.     

Analysis of protein digests by capillary high-performance liquid chromatography and on-line fast atom bombardment mass spectrometry

An HPLC system incorporating a packed capillary C18 column has been utilized for high sensitivity peptide mapping and preparative collection for protein sequencing. This system combined with a Frit-FAB mass spectrometer interface also provides the ability to obtain molecular ions for peptides of enzymatically digested proteins in the time it takes to obtain an HPLC chromatogram. The low flow rates permit introduction of the entire column effluent into the mass spectrometer. Detection limits of 0.5-5 pmol are routine. Proteolytic digests of recombinant human methionyl growth hormone and protein carboxyl methyltransferase have been used to demonstrate the HPLC and mass spectrometer performance.     

Optimization of protein identification from digests as analyzed by capillary isoelectric focusing-mass spectrometry

Capillary isoelectric focusing (CIEF) is a high-resolution separation technique for the analysis of peptides and protein digests. When coupled to ion trap-mass spectrometry (CIEF-MS) the unique separation mechanism is combined with a highly efficient detection system. In an earlier report, we described aspects of separation and interfacing in connection to the analysis of a digest of set of standard proteins. Now, we report on different aspects of the process of protein identification. Sequest software parameters were optimized by using a standard protein digest. These settings were used for the analysis of periplasmic proteins from Escherichia coli. Since in CIEF peptides are focused according to their pI values, the mobilization time of a particular peptide is dependent on its pI value. Based on this relation, the identification of some peptides was facilitated. Furthermore, the Sequest settings that were used could be evaluated. In total, 159 proteins were identified in a single run.     

Global methods for protein glycosylation analysis by mass spectrometry

Mass spectrometry has been an analytical tool of choice for glycosylation analysis of individual proteins. Over the last 5 years several previously and newly developed mass spectrometry methods have been extended to global glycoprotein studies. In this review we discuss the importance of these global studies and the advances that have been made in enrichment analyses and fragmentation methods. We also briefly describe relevant sample preparation methods that have been used for the analysis of a single glycoprotein that could be extrapolated to global studies. Finally this review covers aspects of improvements and advances on the instrument front which are important to future global glycoproteomic studies.     

Single-cell protein analysis by mass spectrometry

Human physiology and pathology arise from the coordinated interactions of diverse single cells. However, analyzing single cells has been limited by the low sensitivity and throughput of analytical methods. DNA sequencing has recently made such analysis feasible for nucleic acids but single-cell protein analysis remains limited. Mass spectrometry is the most powerful method for protein analysis, but its application to single cells faces three major challenges: efficiently delivering proteins/peptides to mass spectrometry detectors, identifying their sequences, and scaling the analysis to many thousands of single cells. These challenges have motivated corresponding solutions, including SCoPE design multiplexing and clean, automated, and miniaturized sample preparation. Synergistically applied, these solutions enable quantifying thousands of proteins across many single cells and establish a solid foundation for further advances. Building upon this foundation, the SCoPE concept will enable analyzing subcellular organelles and posttranslational modifications, while increases in multiplexing capabilities will increase the throughput and decrease cost.     

Characterization of glycoprotein digests with hydrophilic interaction chromatography and mass spectrometry

A new hydrophilic interaction chromatography (HILIC) column packed with amide 1.7 μm sorbent was applied to the characterization of glycoprotein digests. Due to the impact of the hydrophilic carbohydrate moiety, glycopeptides were more strongly retained on the column and separated from the remaining nonglycosylated peptides present in the digest. The glycoforms of the same parent peptide were also chromatographically resolved and analyzed using ultraviolet and mass spectrometry detectors. The HILIC method was applied to glyco-profiling of a therapeutic monoclonal antibody and proteins with several N-linked and O-linked glycosylation sites. For characterization of complex proteins with multiple glycosylation sites we utilized 2D LC, where RP separation dimension was used for isolation of glycopeptides and HILIC for resolution of peptide glycoforms. The analysis of site-specific glycan microheterogeneity was illustrated for the CD44 fusion protein.     

Computational methods for protein identification from mass spectrometry data

Protein identification using mass spectrometry is an indispensable computational tool in the life sciences. A dramatic increase in the use of proteomic strategies to understand the biology of living systems generates an ongoing need for more effective, efficient, and accurate computational methods for protein identification. A wide range of computational methods, each with various implementations, are available to complement different proteomic approaches. A solid knowledge of the range of algorithms available and, more critically, the accuracy and effectiveness of these techniques is essential to ensure as many of the proteins as possible, within any particular experiment, are correctly identified. Here, we undertake a systematic review of the currently available methods and algorithms for interpreting, managing, and analyzing biological data associated with protein identification. We summarize the advances in computational solutions as they have responded to corresponding advances in mass spectrometry hardware. The evolution of scoring algorithms and metrics for automated protein identification are also discussed with a focus on the relative performance of different techniques. We also consider the relative advantages and limitations of different techniques in particular biological contexts. Finally, we present our perspective on future developments in the area of computational protein identification by considering the most recent literature on new and promising approaches to the problem as well as identifying areas yet to be explored and the potential application of methods from other areas of computational biology.     

Microscale affinity purification of trypsin reduces background peptides in matrix-assisted laser desorption/ionization mass spectrometry of protein digests

The use of trypsin for protein digestion is hampered by its autolysis and low thermostability. Chemical modifications have been employed to stabilize the enzyme. Modified trypsin (e.g. methylated) usually enables performing digestions at elevated temperatures, but it still produces autolytic peptides. In this work, unmodified bovine trypsin was subjected to a microscale affinity chromatography on Arginine Sepharose (ASE) or Benzamidine Sepharose (BSE), which utilized the principle of active-site ligand binding. Trypsin was retained on the sorbents in ammonium bicarbonate as a binding buffer. After washings to remove unbound impurities, the enzyme was eluted by arginine as a free ligand (from ASE) or by diluted hydrochloric acid (from BSE). MALDI-TOF mass spectrometry confirmed removal of large molecular fragments as well as autolytic and other background peptides. Consequently, the purified trypsin was tested for its performance in procedures of in-gel digestion of protein standards and selected urinary proteins from real samples. It has been shown that the affinity purification of trypsin decreases significantly the number of unmatched peptides in peptide mass fingerprints. The presence of arginine in the digestion buffer was found to reduce intensity of autolytic peptides. As a result, the described purification procedure is applicable in a common proteomic routine.     

Direct analysis of protein complexes using mass spectrometry.

We describe a rapid, sensitive process for comprehensively identifying proteins in macromolecular complexes that uses multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS/MS) to separate and fragment peptides. The SEQUEST algorithm, relying upon translated genomic sequences, infers amino acid sequences from the fragment ions. The method was applied to the Saccharomyces cerevisiae ribosome leading to the identification of a novel protein component of the yeast and human 40S subunit. By offering the ability to identify >100 proteins in a single run, this process enables components in even the largest macromolecular complexes to be analyzed comprehensively.     

Computational and statistical analysis of protein mass spectrometry data

High-throughput proteomics experiments involving tandem mass spectrometry produce large volumes of complex data that require sophisticated computational analyses. As such, the field offers many challenges for computational biologists. In this article, we briefly introduce some of the core computational and statistical problems in the field and then describe a variety of outstanding problems that readers of PLoS Computational Biology might be able to help solve.     

Selection of affinity ligands for protein purification from proteolytic digests

A simple method for the selection of affinity ligands from proteolytic digests by affinity chromatography is proposed. A small proportion of the peptides in the trypsin digest of bovine serum albumin (BSA) or the pepsin digest of cytochrome are retarded on insulin-immobilised or HSA (human serum albumin)-immobilised affinity columns, respectively. The peptides in these selected fractions can be immobilised onto solid phases and used in affinity chromatography procedures for the purification of insulin or HSA. © Rapid Science Ltd. 1998     

Developments in mass spectrometry for the analysis of complex protein mixtures.

State-of-the-art proteomics workflows involve multiple interdependent steps: sample preparation, protein-peptide separation, mass spectrometry and data analysis. While improvements in any of these steps can increase the depth and breadth of analysis, advances in mass spectrometry have catalysed many of the most important developments. We discuss common classes of mass analysers and how these analysers are put together to produce some of the most popular mass spectrometry platforms. The capabilities of these platforms determine how they can be used in a variety of common proteomic strategies and, in turn, what types of biological questions can be addressed. Moving forward, powerful new hybrid mass spectrometers and application of emerging types of tandem mass spectrometry promise that our ability to analyse complex mixtures of proteins will continue to advance.     

Identification of nearest-neighbor peptides in protease digests by mass spectrometry for construction of sequence-ordered tryptic maps

Continuous-flow fast atom bombardment mass spectrometry was used for the identification of the intermediates and end products of the tryptic digest of polypeptides throughout the time-course of the reactions. Precursor/product relationships for these peptides were determined with the aid of a simple personal computer program. The C-terminal tryptic peptides were identified by performing a tryptic digest in 50% 18O-enriched buffer which resulted in labeling of all non-C-terminal peptides with 18O. This information, along with the precursor/product correlations, was used to create a sequence-ordered tryptic map of the original polypeptide. Ion intensities of intermediate hydrolysis products are compared for enzyme to substrate ratios of 1:100 and 1:1000 (w/w) over the course of the reaction. Intermediates were found to have significantly longer lifetimes when lower levels of trypsin were present.     

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.