Off-line Strong Cation Exchange Research Articles

Identification of Proteomic Biomarkers of Acetaminophen-Induced Hepatotoxicity Using Stable Isotope Labeling.

Quantitative proteomics approaches based on stable isotopic labeling and mass spectrometry have been widely applied to disease research, drug target discovery, biomarker identification, and systems biology. One of the notable stable isotopic labeling approaches is trypsin-catalyzed 18O/16O labeling, which has its own advantages of low sample consumption, simple labeling procedure, cost-effectiveness, and absence of chemical reactions that potentially generate by-products. In this chapter, a protocol for 18O/16O labeling-based quantitative proteomics approach is described with an application to the identification of proteomic biomarkers of acetaminophen (APAP)-induced hepatotoxicity in rats. The protocol involves first the extraction of proteins from liver tissues of control and APAP-treated rats and digestion into peptides by trypsin. After cleaning of the peptides by solid-phase extraction, equal amounts of peptides from the APAP treatment and the control groups are then subject to trypsin-catalyzed 18O/16O labeling. The labeled peptides are combined and fractionated by off-line strong cation exchange liquid chromatography (SCXLC), and each fraction is then analyzed by nanoflow reversed-phase LC coupled online with tandem mass spectrometry (RPLC-MS/MS) for identification and quantification of differential protein expression between APAP-treated rats and controls. The protocol is applicable to quantitative proteomic analysis for a variety of biological samples.

Characterization of Novel Dipeptidyl Peptidase-IV Inhibitory Peptides from Soft-Shelled Turtle Yolk Hydrolysate Using Orthogonal Bioassay-Guided Fractionations Coupled with In Vitro and In Silico Study.

Five novel peptides (LPLF, WLQL, LPSW, VPGLAL, and LVGLPL) bearing dipeptidyl peptidase IV (DPP-IV) inhibitory activities were identified from the gastrointestinal enzymatic hydrolysate of soft-shelled turtle yolk (SSTY) proteins. Peptides were isolated separately using reversed-phase (RP) chromatography in parallel with off-line strong cation exchange (SCX) chromatography followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to determine sequences. Among these peptides, LPSW showed the highest DPP-IV inhibitory activity with an IC50 value of 269.7 ± 15.91 µM. The results of the pre-incubation experiment and the kinetic study of these peptides indicated that WLQL is a true inhibitor and its inhibition toward DPP-IV is of an uncompetitive model, while LPLF, LPSW, and VPGLAL are real-substrates and competitive inhibitors against DPP-IV. The DPP-IV inhibitory peptides derived from SSTY hydrolysate in study are promising in the management of hyperglycemia in Type 2 diabetes.

Exogenous Auxin Elicits Changes in the Arabidopsis thaliana Root Proteome in a Time-Dependent Manner.

Auxin is involved in many aspects of root development and physiology, including the formation of lateral roots. Improving our understanding of how the auxin response is mediated at the protein level over time can aid in developing a more complete molecular framework of the process. This study evaluates the effects of exogenous auxin treatment on the Arabidopsis root proteome after exposure of young seedlings to auxin for 8, 12, and 24 h, a timeframe permitting the initiation and full maturation of individual lateral roots. Root protein extracts were processed to peptides, fractionated using off-line strong-cation exchange, and analyzed using ultra-performance liquid chromatography and data independent acquisition-based mass spectrometry. Protein abundances were then tabulated using label-free techniques and evaluated for significant changes. Approximately 2000 proteins were identified during the time course experiment, with the number of differences between the treated and control roots increasing over the 24 h time period, with more proteins found at higher abundance with exposure to auxin than at reduced abundance. Although the proteins identified and changing in levels at each time point represented similar biological processes, each time point represented a distinct snapshot of the response. Auxin coordinately regulates many physiological events in roots and does so by influencing the accumulation and loss of distinct proteins in a time-dependent manner. Data are available via ProteomeXchange with the identifier PXD001400.

Proteomic profiling of nipple aspirate fluid (NAF): Exploring the complementarity of different peptide fractionation strategies.

High-resolution peptide separation is a sine qua non condition for achieving extensive proteome coverage. Various techniques have been employed to improve peptide fractionation prior to LC-MS/MS, thus allowing a comprehensive characterization of complex biological samples. Although fractionation efficiency is very sample-dependent, this issue is commonly overlooked, and a "cookbook" approach is routinely used during this critical step. The present study provides a systematic comparison of analytical information needed for the successful large-scale differential proteomic analysis of NAF samples from breast cancer patients, a precious and volume-limited biological sample. It reinforces the importance of optimizing sample-specific fractionation protocols for information retrieval from mass spectrometric analysis.

Development of a Novel Cross-linking Strategy for Fast and Accurate Identification of Cross-linked Peptides of Protein Complexes

Knowledge of elaborate structures of protein complexes is fundamental for understanding their functions and regulations. Although cross-linking coupled with mass spectrometry (MS) has been presented as a feasible strategy for structural elucidation of large multisubunit protein complexes, this method has proven challenging because of technical difficulties in unambiguous identification of cross-linked peptides and determination of cross-linked sites by MS analysis. In this work, we developed a novel cross-linking strategy using a newly designed MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). DSSO contains two symmetric collision-induced dissociation (CID)-cleavable sites that allow effective identification of DSSO-cross-linked peptides based on their distinct fragmentation patterns unique to cross-linking types (i.e. interlink, intralink, and dead end). The CID-induced separation of interlinked peptides in MS/MS permits MS(3) analysis of single peptide chain fragment ions with defined modifications (due to DSSO remnants) for easy interpretation and unambiguous identification using existing database searching tools. Integration of data analyses from three generated data sets (MS, MS/MS, and MS(3)) allows high confidence identification of DSSO cross-linked peptides. The efficacy of the newly developed DSSO-based cross-linking strategy was demonstrated using model peptides and proteins. In addition, this method was successfully used for structural characterization of the yeast 20 S proteasome complex. In total, 13 non-redundant interlinked peptides of the 20 S proteasome were identified, representing the first application of an MS-cleavable cross-linker for the characterization of a multisubunit protein complex. Given its effectiveness and simplicity, this cross-linking strategy can find a broad range of applications in elucidating the structural topology of proteins and protein complexes.

High-resolution mass spectrometry proteomics for the identification of candidate plasma protein biomarkers for chronic obstructive pulmonary disease

Although cigarette smoking is recognized as the most important cause of chronic obstructive pulmonary disease (COPD), the pathophysiological mechanisms underlying the lung function decline are not well understood. Using off-line strong cation exchange fractionation with RP-LC-ESI-MS/MS and robust database searching, 1758 tryptic peptides were identified in plasma samples from cigarette smokers. Using two statistical approaches, 30 peptides were identified to be associated with the annualized rate of lung function decline over 5 years among smokers with COPD characterized as having rapid (n = 18) or slow (n = 18) decline and 18 smokers without COPD. The identified peptides belong to proteins that are involved in the complement or coagulation systems or have antiprotease or metabolic functions. This research demonstrates the utility of proteomic profiling to improve the understanding of molecular mechanisms involved in cigarette smoking-related COPD by identifying plasma proteins that correlate with decline in lung function.

Global Proteomic Profiling of Endemic Versus Sporadic Epstein-Barr Virus (EBV) Positive Burkitt Lymphoma (BL).

Abstract 4779 BackgroundEBV infection of normal B-cells is commonly associated with the pathogenesis of BL (Brady et al, Clin Path, 2007). Endemic BL (eBL) is characteristically positive (100%) for EBV, contrasting with sporadic BL (sBL), where approximately 30% of cases are positive for EBV. eBL vs. sBL have significantly different breakpoint regions within c-myc (Shiramizu/Magrath et al, Blood, 1991). Overexpression of c-myc is the sine quae non of BL. C-myc interactions with other genes/proteins is multilayered and complex (Basso/Della-Favera, Nat Gen, 2005). Apoptotic pathway disruption is propelled by EBV and is critically important in c-myc deregulation and subsequent lymphomagenesis that occurs in EBV+ eBL vs. sBL (Ruf et al, J Vir, 2000). Global analysis of proteins expressed in EBV+ eBL vs. sBL may provide insights into biologic, pathogenetic, and molecular differences between the two subtypes of lymphoma, and potentially identify targets for the development of therapeutic agents. ObjectivesTo compare the proteomic expression profile and signal transduction pathways of EBV+ eBL vs. sBL. MethodsWhole cell lysates obtained from the EBV+ eBL cell line Raji and the EBV+ sBL cell line NC37 were digested and labeled with iTRAQ” labeling reagents, following manufacturer's protocol. The peptides were resolved by 2D-LC technique (off-line Strong cation exchange followed by on-line reverse-phase liquid chromatography). Data-dependant High energy C-trap Dissociation MS/MS spectra were acquired using an Orbitrap XL Tandem Mass Spectrometer (ThermoFisher). The MS/MS data was searched using X!Tandem/TPP software suite against human IPI database (v3.50) appended with decoy (reverse) sequences. iTRAQ” ratios of proteins (ProteinProphet probability of >0.9) were normalized and differentially expressed proteins were determined through Mixture Modeling. Protein interactions were further analyzed using the GoMiner and Ingenuity pathway analysis tools. ResultsOver 400 proteins were identified as being differentially expressed by a ≥ 1.25 fold change between the two cell lines. We identified differentially expressed proteins in both cell lines that are involved in a wide array of cellular processes as exhibited in Figure 1. Cellular processes uniquely involved by proteins over-expressed in eBL included immune response, hematopoiesis, cell proliferation, heat shock, and B-cell activation, while those uniquely identified in sBL included cell division, response to virus, and NF-kB cascade proteins. Specific cell-regulatory pathways implicated by the differential protein profile expressions (with associated proteins in parentheses) included the p53 apoptosis pathway (PCNA, MSH6, C1QBP, MAP4, and BAX), the caspase network of apoptosis (HCLS1, ACIN1, and AIFM1), the tumor suppressor protein RB network (MCM7, PA2G4, and API5), general apoptotic pathways (HSP90 and PDCD4), B-cell differentiation and proliferation pathways (TPD52 and IKBKG), and the ubiquitin-proteasome pathway (UBE2J1, UBE2C, and UBE2S). Seven of these proteins are c-myc target genes. Ingenuity protein network analysis revealed nine proteins identified in the experiment with interactions connected through the p53, caspase, and tumor necrosis factor apoptosis pathways. ConclusionProteomic profile analysis of EBV+ eBL and sBL revealed over and under-expression of multiple proteins that may be implicated in the multi-factorial nature of disease pathogenesis. This is the first MS-based direct proteomic comparison of eBL and sBL. Our results suggest that there are potentially different mechanisms driving cell proliferation and resistance to apoptosis in eBL versus sBL and that EBV infection may be involved in the processes that drive lymphomagenesis. Ultimately, identification of proteins unique to the distinct disease subtypes will serve to establish tumor markers that may enable development of new diagnostic, prognostic, and therapeutic strategies. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

An Efficient Organic Solvent Based Extraction Method for the Proteomic Analysis ofArabidopsisPlasma Membranes

Membrane proteins are involved in diverse cellular processes and are an integral component of many signaling cascades, but due to their highly hydrophobic nature and the complexities associated with studying these proteins in planta, alternative methods are being developed to better characterize these proteins on a proteome-wide scale. In our previous work ( Mitra , S. K. et al. J. Proteome Res. 2007 , 6 , ( 5 ), 1933 - 50 ), methanol-assisted solubilization was determined to facilitate the identification of both hydrophobic and hydrophilic membrane proteins compared to Brij-58 solubilization and was particularly effective for leucine-rich repeat receptor-like kinases (LRR RLKs). To improve peptide identification and to overcome sample losses after tryptic digestion, we have developed an effective chloroform extraction method to promote plasma membrane protein identification. The use of chloroform extraction over traditional solid-phase extraction (SPE) prior to off-line strong cation exchange liquid chromatography (SCXC) and reversed-phase liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis facilitated the removal of chlorophylls, major contaminants of plant tissue preparations that can affect downstream analysis, in addition to the effective removal of trypsin used in the digestion. On the basis of a statistically derived 5% false discovery rate, the chloroform extraction procedure increased the identification of unique peptides for plasma membrane proteins over SPE by 70% which produced nearly a 2-fold increase in detection of membrane transporters and LRR RLKs without increased identification of contaminating Rubisco and ribosomal peptides. Overall, the combined use of methanol and chloroform provides an effective method to study membrane proteins and can be readily applied to other tissues and cells types for proteomic analysis.

Optimizing the peak capacity per unit time in one-dimensional and off-line two-dimensional liquid chromatography for the separation of complex peptide samples

To obtain the best compromise between peak capacity and analysis time in one-dimensional and two-dimensional (2D) liquid chromatography (LC), column technology and operating conditions were optimized. The effects of gradient time, flow rate, column temperature, and column length were investigated in one-dimensional reversed-phase (RP) gradient nano-LC, with the aim of maximizing the peak per unit time for peptide separations. An off-line two-dimensional LC approach was developed using a micro-fractionation option of the autosampler, which allowed automatic fractionation of peptides after a first-dimension ion-exchange separation and re-injection of the fractions onto a second-dimension RP nano-LC column. Under the applied conditions, which included a preconcentration/desalting time of 5 min, and a column equilibration time of 12.5 min, the highest peak capacity per unit time in the 2D-LC mode was obtained when applying a short (10 min) first-dimension gradient and second-dimension RP gradients of 20 min duration. For separations requiring a maximum peak capacity of 375, one-dimensional LC was found to be superior to the off-line strong cation-exchange/×/RPLC approach in terms of analysis time. Although a peak capacity of 450 could be obtained in one-dimensional LC when applying 120-min gradients on 500-mm long columns packed with 3-μm particles, for separations requiring a peak capacity higher than 375 2D-LC experiments provide a higher peak capacity per unit time. Finally, the potential of off-line 2D-LC coupled to tandem mass spectrometry detection is demonstrated with the analysis of a tryptic digest of a mixture of nine proteins and an Escherichia coli digest.

Analysis of human liver proteome using replicate shotgun strategy

In this study, a liquid-based shotgun strategy was used to comprehensively identify the expression of human liver proteome. Proteins were extracted from human liver tissue and digested in-solution. The tryptic digest mixture was desalted and separated by off-line strong cation exchange (SCX) chromatography with a 60-min elution. The MS/MS spectra were acquired in data-dependent mode after an RP chromatographic separation combined with linear IT MS analysis. To obtain the most comprehensive human liver proteome, each SCX fraction was run six times in RPLC MS/MS manner. Finally, more than 6,000,000 MS/MS spectra were collected. Using a relatively strict filter criteria, 24,311 proteins (48.42% of the predicted human proteome from human International Protein Index (IPI) protein database 3.07) corresponding to 13,150 nonredundant proteins were successfully identified, in which 7001 proteins (53.24%) were identified by two or more peptides, which could be considered as a high-confident dataset. Among the 6149 proteins (46.76%) identified by single peptide, 3812 proteins (61.99%) were detected more than twice in six repeated runs. Comparative analysis between different runs shows that the overlap of identified proteins between any two runs ranged from 25 to 44%. Of the nonredundant proteins identified, 8919 proteins (67.83%) were detected more than twice and 4231 proteins (32.17%) were detected only once in six RPLC MS/MS runs. The Gene Ontology annotation shows that the identified proteins come from various subcellular components. In addition, a large number of low abundant proteins were identified. The dynamic range of the approach reached at least nine orders of magnitude by estimating the concentration of proteins.

Improved Validation of Peptide MS/MS Assignments Using Spectral Intensity Prediction

A major limitation in identifying peptides from complex mixtures by shotgun proteomics is the ability of search programs to accurately assign peptide sequences using mass spectrometric fragmentation spectra (MS/MS spectra). Manual analysis is used to assess borderline identifications; however, it is error-prone and time-consuming, and criteria for acceptance or rejection are not well defined. Here we report a Manual Analysis Emulator (MAE) program that evaluates results from search programs by implementing two commonly used criteria: 1) consistency of fragment ion intensities with predicted gas phase chemistry and 2) whether a high proportion of the ion intensity (proportion of ion current (PIC)) in the MS/MS spectra can be derived from the peptide sequence. To evaluate chemical plausibility, MAE utilizes similarity (Sim) scoring against theoretical spectra simulated by MassAnalyzer software (Zhang, Z. (2004) Prediction of low-energy collision-induced dissociation spectra of peptides. Anal. Chem. 76, 3908-3922) using known gas phase chemical mechanisms. The results show that Sim scores provide significantly greater discrimination between correct and incorrect search results than achieved by Sequest XCorr scoring or Mascot Mowse scoring, allowing reliable automated validation of borderline cases. To evaluate PIC, MAE simplifies the DTA text files summarizing the MS/MS spectra and applies heuristic rules to classify the fragment ions. MAE output also provides data mining functions, which are illustrated by using PIC to identify spectral chimeras, where two or more peptide ions were sequenced together, as well as cases where fragmentation chemistry is not well predicted.

Lumbar Cerebrospinal Fluid Proteome in Multiple Sclerosis: Characterization by Ultrafiltration, Liquid Chromatography, and Mass Spectrometry

Neurological diseases, including multiple sclerosis (M.S.), often provoke changes in the functioning of the endothelial and epithelial brain barriers and give rise to disease-associated alterations of the cerebrospinal fluid (CSF) proteome. In the present study, pooled and ultrafiltered CSF of M.S. and non-M.S. patients was digested with trypsin and analyzed by off-line strong cation-exchange chromatography (SCX) coupled to on-line reversed-phase LC-ESI-MS/MS. In an alternative approach, the trypsin-treated subproteomes were analyzed directly by LC-ESI-MS/MS and gas-phase fractionation in the mass spectrometer. Taken together, both proteomic approaches in combination with a three-step evaluation process including the search engines Sequest and Mascot, and the validation software Scaffold, resulted in the identification of 148 proteins. Sixty proteins were identified in CSF for the first time by mass spectrometry. For validation purposes, the concentration of cystatin A was determined in individual CSF and serum samples of M.S. and non-M.S. patients using ELISA.

Multi‐dimensional HPLC/MS of the nucleolar proteome using HPLC‐chip/MS

The proteome of the human nucleolus was investigated in a single analysis using off-line strong cation exchange chromatography and microfraction collection combined with HPLC-chip/MS. The analysis was conducted either as a 1-D workflow with HPLC-chip alone or as a 2-D workflow. Two hundred and six unique proteins were identified in the International Protein Index human database corresponding to 2024 unique tryptic peptides identified in the 2-D analysis. In contrast, only 34 proteins and 151 corresponding tryptic peptides were found by applying a 1-D separation strategy. This clearly indicated that the complexity of the samples required the combination of more than one orthogonal separation technique. Stringent database search criteria, including reversal of sequences and therefore better exclusion of false-positive identifications, were applied for reliable protein identification.

The Turnover Kinetics of Major Histocompatibility Complex Peptides of Human Cancer Cells

Peptides presented by the major histocompatibility complex (MHC) are derived from the degradation of cellular proteins. Thus, the repertoire of these peptides (the MHC peptidome) should correlate better with the cellular protein degradation scheme (the degradome) than with the cellular proteome. To test the validity of this statement and to determine whether the majority of MHC peptides are derived from short lived proteins, from defective ribosome products, or from regular long lived cellular proteins we analyzed in parallel the turnover kinetics of both MHC peptides and cellular proteins in the same cancer cells. The analysis was performed by pulse-chase experiments based on stable isotope labeling in tissue culture followed by capillary chromatography and tandem mass spectrometry. Indeed only a limited correlation was observed between the proteome and the MHC peptidome observed in the same cells. Moreover a detailed analysis of the turnover kinetics of the MHC peptides helped to assign their origin to normal, to short lived or long lived proteins, or to the defective ribosome products. Furthermore the analysis of the MHC peptides turnover kinetics helped to direct attention to abnormalities in the degradation schemes of their source proteins. These observations can be extended to search for cancer-related abnormalities in protein degradation, including those that lead to loss of tumor suppressors and cell cycle regulatory proteins.

Protein expression profiling of CLL B cells using replicate off-line strong cation exchange chromatography and LC–MS/MS

In this study we use replicate 2D-LC–MS/MS analyses of crude membranes from B cells derived from a patient with chronic lymphocytic leukemia (CLL) to examine the protein expression profile of CLL B cells. Protein identifications made by replicate 2D-LC–MS/MS analysis of tryptic peptides from detergent solubilized B cell membrane proteins, as well as replicate LC–MS/MS analysis of single off-line strong cation exchange chromatography (SCX) fractions, were analyzed. We show that despite the variance in SCX, capillary LC, and the data-dependent selection of precursor ions, an overlap of 64% between proteins identified in replicate runs was achieved for this system.

Proteome Changes Induced by Rituximab: Significance for Monoclonal Antibody Therapy of B-Cell Lymphoproliferative Disorders.

Rituximab is a human chimeric monoclonal antibody that targets the CD20 receptor uniquely expressed on normal and malignant B-cells. While it is particularly useful for follicular lymphoma, Rituximab has recently been used for other types of B-cell proliferative disorders. Four major mechanisms have been proposed to account for the activity of Rituximab in B-cell lymphoproliferative disorders: 1) apoptosis and proliferation inhibition via intracellular signaling, 2) increased sensitivity to other chemotherapeutic drugs, 3) complement-dependent cytotoxicity (CDC), and 4) antibody-dependent cellular cytotoxicity (ADCC). A combination of all four mechanisms has also been proposed as the mechanism of action. However, the intracellular events that occur in B-cells in response to Rituximab are largely unknown.We have employed a global quantitative proteomic profiling approach to study the effects of Rituximab on a t(14;18)+ B cell lymphoma (SUDHL-4). A decrease in viability of 31.3 ± 2.9% was observed after treatment with 10μg/mL of Rituximab for 48 hours. We used isotope-coded affinity tagging (ICAT™) in combination with microcapillary liquid chromatography and tandem mass spectrometry (μLC-MS/MS) to quantitatively determine the proteomic differences between equal amounts of untreated and treated total cell lystates. Protein lysates were labeled with either heavy or light cleavable ICAT™ reagents, digested with trypsin, and purified using avidin affinity chromatography. The labeled peptides were then fractionated using off-line strong cation exchange (SCX) and each fraction was subjected to μLC-MS/MS analysis. Several proteins previously unknown to be expressed in B-cells were identified using this approach. Of the 123 positively identified proteins, 59 proteins were differentially expressed by greater than or less than 1.5 fold (25 upregulated, 34 downregulated). Analysis of differentially expressed proteins identified several functional groups, including integral membrane proteins (Integrin alpha-7 precursor), transcription factors (Zinc finger proteins 445 and 20), proteins involved in migration and adhesion (Astrotactin1), proteins associated with degradation and turnover (26S proteasome non-ATPase regulatory subunit 9), proteins affecting calcium-induced signaling (Tumor-associated calcium signal transducer 1 precursor), proteins associated with lipid rafts (Gemin7), and components of the phosphoinositol (PI4K-alpha) and NF-κB pathways (IKK-E). Our studies reveal the proteomic consequences of Rituximab administration to neoplastic B-cells, and provide novel insights into the mechanism of the drug's efficacy in the treatment of susceptible B-cell lymphoproliferative disorders.