In-Gel Stable-Isotope Labeling (ISIL): a strategy for mass spectrometry-based relative quantification

Most proteomics approaches for relative quantification of protein expression use a combination of stable-isotope labeling and mass spectrometry. Traditionally, researchers have used difference gel electrophoresis (DIGE) from stained 1D and 2D gels for relative quantification. While differences in protein staining intensity can often be visualized, abundant proteins can obscure less abundant proteins, and quantification of post-translational modifications is difficult. A method is presented for quantifying changes in the abundance of a specific protein or changes in specific modifications of a protein using In-gel Stable-Isotope Labeling (ISIL). Proteins extracted from any source (tissue, cell line, immunoprecipitate, etc.), treated under two experimental conditions, are resolved in separate lanes by gel electrophoresis. The regions of interest (visualized by staining) are reacted separately with light versus heavy isotope-labeled reagents, and the gel slices are then mixed and digested with proteases. The resulting peptides are then analyzed by LC-MS to determine relative abundance of light/heavy isotope pairs and analyzed by LC-MS/MS for identification of sequence and modifications. The strategy compares well with other relative quantification strategies, and in silico calculations reveal its effectiveness as a global relative quantification strategy. An advantage of ISIL is that visualization of gel differences can be used as a first quantification step followed by accurate and sensitive protein level stable-isotope labeling and mass spectrometry-based relative quantification.     

In-gel stable isotope labeling for relative quantification using mass spectrometry

Although differences in protein staining intensity can often be visualized by difference gel electrophoresis, abundant proteins can obscure less abundant proteins, and quantification of post-translational modifications is difficult. We present a protocol for quantifying changes in the abundance of a specific protein or changes in specific modifications of a protein using in-gel stable isotope labeling. In this protocol protein extracts from any source treated under two experimental conditions are resolved in two separate lanes by gel electrophoresis. Parallel gel regions of interest are reacted separately with either light or heavy isotope-labeled reagents, and the gel slices are then combined and digested with proteases. The resulting peptides are then analyzed by liquid chromatography/mass spectrometry (LC/MS) to determine relative abundance of light- and heavy-isotope lysine-containing peptide pairs and analyzed by LC/MS/MS for identification of sequence and modifications. This protocol should take approximately 24-26 h to complete, including the incubation time for proteolytic digestion. Additional time will be needed for data analysis and interpretation.     

Gel-free mass spectrometry-based high throughput proteomics: tools for studying biological response of proteins and proteomes

Revolutionary advances in biological mass spectrometry (MS) have provided a basic tool to make possible comprehensive proteomic analysis. Traditionally, two-dimensional gel electrophoresis has been used as a separation method coupled with MS to facilitate analysis of complex protein mixtures. Despite the utility of this method, the many challenges of comprehensive proteomic analysis has motivated the development of gel-free MS-based strategies to obtain information not accessible using two-dimensional gel separations. These advanced strategies have enabled researchers to dig deeper into complex proteomes, gaining insights into the composition, quantitative response, covalent modifications and macromolecular interactions of proteins that collectively drive cellular function. This review describes the current state of gel-free, high throughput proteomic strategies using MS, including (i) the separation approaches commonly used for complex mixture analysis; (ii) strategies for large-scale quantitative analysis; (iii) analysis of post-translational modifications; and (iv) recent advances and future directions. The use of these strategies to make new discoveries at the proteome level into the effects of disease or other cellular perturbations is discussed in a variety of contexts, providing information on the potential of these tools in electromagnetic field research.     

Sub-proteome differential display: single gel comparison by 2D electrophoresis and mass spectrometry

Two-dimensional (2D) gel electrophoresis and mass spectrometry (MS) have been used in comparative proteomics but inherent problems of the 2D electrophoresis technique lead to difficulties when comparing two samples. We describe a method (sub-proteome differential display) for comparing the proteins from two sources simultaneously. Proteins from one source are mixed with radiolabelled proteins from a second source in a ratio of 100:1. These combined proteomes are fractionated simultaneously using column chromatographic methods, followed by analysis of the pre-fractionated proteomes (designated sub-proteomes) using 2D gel electrophoresis. Silver staining and (35)S autoradiography of a single gel allows precise discrimination between members of each sub-proteome, using commonly available computer software. This is followed by MS identification of individual proteins. We have demonstrated the utility of the technology by identifying the product of a transfected gene and several proteins expressed differentially between two renal carcinoma proteomes. The procedure has the capacity to enrich proteins prior to 2D electrophoresis and provides a simple, inexpensive approach to compare proteomes. The single gel approach eliminates differences that might arise if separate proteome fractionations or 2D gels are employed.     

The role of mass spectrometry in proteome studies

Mass spectrometry (MS) is an important tool in modern protein chemistry. In proteome analyses the expression of hundreds or thousands of proteins can be monitored at the same time. First, complex protein mixtures are separated by two-dimensional gel electrophoresis (2-DE) and then individual proteins are identified by using MS followed by database searches. Recent developments in this field have made it possible to do automated, high-throughput protein identification that is needed in proteome analyses. MS can also be used to characterize post-translational modifications in proteins and to study protein complexes. This review will introduce the current MS methods used in proteome studies, and discuss their advantages and disadvantages. New instrumental MS developments are also presented that are useful in these analyses.     

Strategies for revealing lower abundance proteins in two-dimensional protein maps

One of the most challenging contemporary research endeavors is the mapping of proteins and establishing their linkages to normal and pathological conditions. The availability of current proteomics technologies has greatly facilitated the separation and identification of proteins in a complex protein mixture by standard two-dimensional gel electrophoresis and subsequent MALDI-TOF mass spectrometry. Due to the huge differences in the distribution of proteins in complex proteomes of humans, the detection and identification of proteins expressed in low copy number is a major challenge. The low abundance of important physiologically relevant proteins has rendered their analyses almost impossible without some means of prior purification and enrichment from tissue lysates or biological fluids. It is the current limits of detection of the methods that are used that prevents the detection of these proteins not the proteins themselves. More importantly, considering the frequency at which post-translational modifications of proteins occur, the separation of protein isoforms is essential to understand biological changes, and two-dimensional gel electrophoresis remains the only technique that can offer sufficient resolution to address this issue at a functional level. Cellular fractionation techniques followed by specific affinity probes for tracking target proteins have been developed to deplete the proteome of high abundance proteins in order to increase the sample loading for achieving greater sensitivity for proteins present in low abundance. Those applications can entail the removal of one protein or a class of proteins that interferes with the resolution of proteins in a 2-DE map. Moreover, the use of better solubilizing detergents in combination with an overlapping narrow immobilized pH gradients, results in higher resolution by stretching the protein pattern in the first dimension. In this review we will discuss strategies to remove high abundance proteins that can result in the visualization and detection of low abundance proteins in biological samples. The potential use of these strategies, as a means of developing diagnostic tools for early screening of diseases and identification of drug targets for therapeutic intervention, will also be discussed.     

Tube-gel digestion: a novel proteomic approach for high throughput analysis of membrane proteins

This study describes a new protein digestion protocol in which a variety of detergents can be used to solubilize membrane proteins and facilitate trypsin digestion with higher efficiency. In this protocol, proteins are dissolved in solutions containing various detergents and directly incorporated into a polyacrylamide gel matrix without electrophoresis. Detergents are subsequently eliminated from the gel matrix while proteins are still immobilized in the gel matrix. After in-gel digestion of proteins, LC-MS/MS is used to analyze the extracted peptides for protein identification. The uniqueness of the protocol is that it allows usage of a variety of detergents in the starting solution without interfering with LC-MS/MS analysis. We hereby demonstrate that different detergents, including ionic SDS, non-ionic Triton X-100 and n-octyl beta-d-glucopyranoside, and zwitterionic CHAPS, can be used to achieve maximum solubilization of membrane proteins with minimal interference with LC-MS/MS analysis. Enhanced digestions, i.e. improved number and intensity of detected peptides, are also demonstrated for digestion-resistant proteins such as myoglobin, ubiquitin, and bacteriorhodopsin. An additional advantage of the Tube-Gel digestion protocol is that, even without electrophoresis separation, it allows high throughput analysis of complex protein mixtures when coupled with LC-MS/MS. The protocol was used to analyze a complex membrane protein mixture prepared from prostate cancer cells. The protocol involves only a single digestion and 2.5 h of LC-MS/MS analysis and identified 178 membrane proteins. In comparison, the same membrane fraction was resolved by SDS-PAGE, and 20 gel slices were excised and individually digested and analyzed by LC-MS/MS. The more elaborate effort demanded more than 50 h of LC-MS/MS analysis and identified 268 proteins. The new Tube-Gel digestion protocol is an alternative method for high throughput analysis of membrane proteins.     

Improved molecular weight-based processing of intact proteins for interrogation by quadrupole-enhanced FT MS/MS

Complete coverage of protein primary structure is demonstrated for 37 yeast protein forms between 6 and 30 kDa in an improved platform for Top Down mass spectrometry (MS). Tandem mass spectrometry (MS/MS) for protein identification with 100% sequence coverage is achieved in a highly automated fashion with 15-300-fold less sample amounts than an initial report of a proteome fractionation approach employing preparative gel electrophoresis with an acid-labile surfactant to facilitate reversed phase separation in a second dimension. Using a quadrupole-enhanced Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FTICRMS) improves the dynamic range for protein detection by approximately 50-fold and MS/MS by approximately 30-fold. The technology development illustrated here typifies an accelerating effort to detect whole proteins in a more general and higher throughput fashion for improved biomarker identification and detection of diverse post-translational modifications. Capillary RPLC is used in both off-line and on-line modes, with one on-line LC/FTMS sample providing 25 observed protein forms from 11 to 22 kDa.     

Protein profiling by the combination of two independent mass spectrometry techniques

Protein profiling in the high-throughput mode is a most useful technique that allows formation of reference databases for cells and tissues and performance of comparative proteomics. In the proposed protocol protein extraction from tissues is followed by 2D gel electrophoresis (2DE) with subsequent in-gel digestion and identification of soluble proteins by two individual mass spectrometric techniques, tandem matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and nano-liquid chromatography (nano-LC)-MS/MS. The proposed combined use of these two MS approaches leads to a very high identification rate of well-separated protein spots from a gel. In the first step 2DE separates high-abundance proteins (those visualized by nonsensitive Coomassie blue staining) that are subsequently picked, digested and aliquoted for MS applications. Protein samples not identified by MALDI-MS or MS/MS (77% of all spots) are finally unambiguously identified by nano-LC-MS/MS (total identification rate 94%). This protocol can be completed in 6 weeks.     

Proteomic Screening for Amyloid Proteins

Despite extensive study, progress in elucidation of biological functions of amyloids and their role in pathology is largely restrained due to the lack of universal and reliable biochemical methods for their discovery. All biochemical methods developed so far allowed only identification of glutamine/asparagine-rich amyloid-forming proteins or proteins comprising amyloids that form large deposits. In this article we present a proteomic approach which may enable identification of a broad range of amyloid-forming proteins independently of specific features of their sequences or levels of expression. This approach is based on the isolation of protein fractions enriched with amyloid aggregates via sedimentation by ultracentrifugation in the presence of strong ionic detergents, such as sarkosyl or SDS. Sedimented proteins are then separated either by 2D difference gel electrophoresis or by SDS-PAGE, if they are insoluble in the buffer used for 2D difference gel electrophoresis, after which they are identified by mass-spectrometry. We validated this approach by detection of known yeast prions and mammalian proteins with established capacity for amyloid formation and also revealed yeast proteins forming detergent-insoluble aggregates in the presence of human huntingtin with expanded polyglutamine domain. Notably, with one exception, all these proteins contained glutamine/asparagine-rich stretches suggesting that their aggregates arose due to polymerization cross-seeding by human huntingtin. Importantly, though the approach was developed in a yeast model, it can easily be applied to any organism thus representing an efficient and universal tool for screening for amyloid proteins.     

Nonredundant mass spectrometry: a strategy to integrate mass spectrometry acquisition and analysis

Protein identification using automated data-dependent tandem mass spectrometry (MS/MS) is now a standard procedure. However, in many cases data-dependent acquisition becomes redundant acquisition as many different peptides from the same protein are fragmented, whilst only a few are needed for unambiguous identification. To increase the quality of information but decrease the amount of information, a nonredundant MS (nrMS) strategy has been developed. With nrMS, data analysis is an integral part of the overall MS acquisition and analysis, and not an endpoint as typically performed. In this nrMS workflow a matrix assisted laser desorption/ionization-time of flight-time of flight (MALDI-TOF/TOF) instrument is used. MS and restricted MS/MS data are searched and identified proteins are used to generate an "exclusion list", after in silico digestion. Peptide fragmentation is then restricted to only the most intense ions not present in the exclusion list. This process is repeated until all peaks are accounted for or the sample is consumed. Compared to nanoLC-MS/MS, nrMS yielded similar results for the analysis of six pooled two-dimensional electrophoresis (2-DE) spots. In comparison to standard data-dependent MALDI-MS/MS for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel band analysis, nrMS dramatically increased the number of identified proteins. It was also found that this new workflow significantly increased sequence coverage by identifying unexpected peptides, which can result from post-translational modifications.     

High resolution mass spectrometric alveolar proteomics: identification of surfactant protein SP-A and SP-D modifications in proteinosis and cystic fibrosis patients

In the present study, one- and two-dimensional gel electrophoresis combined with high resolution Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) have been applied as powerful approaches for the proteome analysis of surfactant proteins SP-A and SP-D, including identification of structurally modified and truncation forms, in bronchoalveolar lavage fluid from patients with cystic fibrosis, chronic bronchitis and pulmonary alveolar proteinosis. Highly sensitive micropreparation techniques were developed for matrix-assisted laser desorption/ionization (MALDI) FT-ICR MS analysis which provided the identification of surfactant proteins at very low levels. Owing to the high resolution, FT-ICR MS was found to provide substantial advantages for the structural identification of surfactant proteins from complex biological matrices with high mass determination accuracy. Several protein bands corresponding to SP-A and SP-D were identified by MALDI-FT-ICR MS after electrophoretic separation by one- and two-dimensional gel electrophoresis, and provided the identification of structural modifications (hydroxy-proline) and degradation products. The high resolution mass spectrometric proteome analysis should facilitate the unequivocal identification of subunits, aggregations, modifications and degradation products of surfactant proteins and hence contribute to the understanding of the mechanistic basis of lung disease pathogenesis.     

Functional consequences of actin nitration: in vitro and in disease states

To link the phenomena of inflammatory-induced increases in protein nitrotyrosine (NO₂Tyr) derivatives to protein dysfunction and consequent pathological conditions, the evaluation of discrete NO₂Tyr modifications on specific proteins must be undertaken. Mass spectrometric (MS) proteomics-based strategies allow for the identification of all individual proteins that are nitrated by separating tissue homogenates using 2D gel electrophoresis, detecting the nitrated proteins using an anti-NO₂Tyr antibody, and then identifying the peptides generated during an in-gel proteolytic digest using matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) MS. Actin, one of the most abundant proteins in eukaryotic cells, constitutes 5% or more of cell protein and serves with other cytoskeletal proteins as a critical target for nitration-induced functional impairment. Herein, examples of actin nitration detected under physiological conditions in various models of human disease or in clinically derived tissues are given and the impact that this post-translational protein modification can have on cell and organ function is discussed.     

Proteomic changes associated with diabetes in the BB-DP rat

These studies were structured with the aim of utilizing emerging technologies in two-dimensional (2D) gel electrophoresis and mass spectrometry to evaluate protein expression changes associated with type 1 diabetes. We reasoned that a broad examination of diabetic tissues at the protein level might open up novel avenues of investigation of the metabolic and signaling pathways that are adversely affected in type 1 diabetes. This study compared the protein expression of the liver, heart, and skeletal muscle of diabetes-prone rats and matched control rats by semiquantitative liquid chromatography-mass spectrometry and differential in-gel 2D gel electrophoresis. Differential expression of 341 proteins in liver, 43 in heart, and 9 (2D gel only) in skeletal muscle was detected. These data were assembled into the relevant metabolic pathways affected primarily in liver. Multiple covalent modifications were also apparent in 2D gel analysis. Several new hypotheses were generated by these data, including mechanisms of net cytosolic protein oxidation, formaldehyde generation by the methionine cycle, and inhibition of carbon substrate oxidation via reduction in citrate synthase and short-chain acyl-CoA dehydrogenase.     

Artifactual sulfation of silver-stained proteins: implications for the assignment of phosphorylation and sulfation sites

Sulfation and phosphorylation are post-translational modifications imparting an isobaric 80-Da addition on the side chain of serine, threonine, or tyrosine residues. These two post-translational modifications are often difficult to distinguish because of their similar MS fragmentation patterns. Targeted MS identification of these modifications in specific proteins commonly relies on their prior separation using gel electrophoresis and silver staining. In the present investigation, we report a potential pitfall in the interpretation of these modifications from silver-stained gels due to artifactual sulfation of serine, threonine, and tyrosine residues by sodium thiosulfate, a commonly used reagent that catalyzes the formation of metallic silver deposits onto proteins. Detailed MS analyses of gel-separated protein standards and Escherichia coli cell extracts indicated that several serine, threonine, and tyrosine residues were sulfated using silver staining protocols but not following Coomassie Blue staining. Sodium thiosulfate was identified as the reagent leading to this unexpected side reaction, and the degree of sulfation was correlated with increasing concentrations of thiosulfate up to 0.02%, which is typically used for silver staining. The significance of this artifact is discussed in the broader context of sulfation and phosphorylation site identification from in vivo and in vitro experiments.     

Identification of virulence factors and diagnostic markers using immunosecretome of Aspergillus fumigatus

Aspergillus fumigatus is a prime causative agent for various allergic and invasive aspergillosis. There has been a dramatic increase of such cases in last three decades yet the early diagnosis and virulence factor identification remains the challenge. In the present study secretome analysis of proteins isolated from the culture filtrate was done by 2D gel electrophoresis coupled with MS/MS and the immunosecretome analysis was carried out using immunoblotting of 2D transfer blots and probed with the sera of patients, immunized rabbit and mice. The identified proteins were analyzed further for homology with human proteins by BLAST search and for secretory signal by SignalP. A total of 65 protein spots from 2D gel resulted in identification of 24 different proteins along with their isoforms and out of which 15 proteins were identified as immunogenic in human. These findings may be helpful in the identification of virulence factors involved in aspergillosis and also useful as diagnostic markers.     

Cancer immunomics using autoantibody signatures for biomarker discovery

The increased incidence of autoantibodies in malignancies has been described since the 1970s. Thus the ability to determine molecular fingerprinting of autoantibodies (antibody signatures) may provide useful clinical diagnostic and prognostic information. This review describes the use of several proteomics approaches for the identification of antigens recognized by these autoantibodies. Serological proteome analysis combines separation of tumor cell proteins on two-dimensional gel electrophoresis gels, Western blotting with sera of patients and healthy subjects, and identification of the detected antigens by MS. Alternatively multiple affinity protein profiling combines isolation of the antigens recognized by patient antibodies by two-dimensional immunoaffinity chromatography and identification by MS/MS. The use and limitations of reverse phase protein microarrays for testing patient serum containing autoantibodies are also considered. Lastly the most important difficulty of any proteomically identified autoantibody signature is validation in patient cohorts or clinical samples.     

Ocular Proteomics with Emphasis on Two-Dimensional Gel Electrophoresis and Mass Spectrometry

The intention of this review is to provide an overview of current methodologies employed in the rapidly developing field of ocular proteomics with emphasis on sample preparation, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Appropriate sample preparation for the diverse range of cells and tissues of the eye is essential to ensure reliable results. Current methods of protein staining for 2D-PAGE, protein labelling for two-dimensional difference gel electrophoresis, gel-based expression analysis and protein identification by MS are summarised. The uses of gel-free MS-based strategies (MuDPIT, iTRAQ, ICAT and SILAC) are also discussed. Proteomic technologies promise to shed new light onto ocular disease processes that could lead to the discovery of strong novel biomarkers and therapeutic targets useful in many ophthalmic conditions.     

Ocular proteomics with emphasis on two-dimensional gel electrophoresis and mass spectrometry

The intention of this review is to provide an overview of current methodologies employed in the rapidly developing field of ocular proteomics with emphasis on sample preparation, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Appropriate sample preparation for the diverse range of cells and tissues of the eye is essential to ensure reliable results. Current methods of protein staining for 2D-PAGE, protein labelling for two-dimensional difference gel electrophoresis, gel-based expression analysis and protein identification by MS are summarised. The uses of gel-free MS-based strategies (MuDPIT, iTRAQ, ICAT and SILAC) are also discussed. Proteomic technologies promise to shed new light onto ocular disease processes that could lead to the discovery of strong novel biomarkers and therapeutic targets useful in many ophthalmic conditions.