Nano-reversed Phase Liquid Research Articles

Cloud-Point Extraction and Delipidation of Porcine Brain Proteins in Combination with Bottom-Up Mass Spectrometry Approaches for Proteome Analysis

In this study, temperature-induced phase fractionation also known as cloud-point extraction (CPE) with the nonionic surfactant Triton X-114 was used to simultaneously extract hydrophobic and hydrophilic proteins from porcine brain tissue. Various protein precipitation/delipidation procedures were investigated to efficiently remove lipids and detergents while retaining maximum protein recoveries. The best performing delipidation method was then used in combination with CPE to compare three different mass spectrometry (MS) based "bottom-up" proteomic approaches for protein analysis of the porcine brain. In the first approach, the intact proteins were initially separated by one-dimensional (1D) gel electrophoresis. The excised protein bands were digested with trypsin, and the peptides were separated by reversed phase nanoliquid chromatography (RP-nanoLC) followed by electrospray ionization (ESI) tandem mass spectrometry (MS/MS) analysis. The other bottom-up proteomic approaches were based on first enzymatical digestion of the proteins followed by RP-nanoLC separation in combination with matrix assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) or on the combination of in-solution isoelectric focusing (IEF) with ESI-nanoLC-MS/MS of the IEF separated peptides. In total, we found and unambiguously identified 331 unique proteins. The overlap between different techniques was about 10%, showing that the use of multiple proteomic approaches is beneficial to yield a better coverage of the proteome. Furthermore, the overlap between the CPE extracted hydrophilic and hydrophobic proteins was rather small (9-16%), indicating an efficient sample preparation technique to extract and separate hydrophilic and hydrophobic proteins from brain tissue. The percentage of identified membrane proteins was 27%, which is in accordance to the fact that about one-third of all genes in various organisms encode for this class of proteins. The results indicate that cloud point extraction is a promising sample preparation tool, which allows simultaneous in depth studies of brain derived membrane proteins as well as hydrophilic proteins. This technique can be very useful when studying human central nervous system (CNS) tissue or animal models of neurological diseases.

Abstract 4569: Serum proteome analysis by label-free quantification system and LC-MALDI mass spectrometry for the discovery of novel biomarkers for lung cancer

Abstract Lung cancer is the leading cause of cancer death in Japan and throughout the world, and highly specific and sensitive serum biomarkers applicable for screening are urgently required. Here we report our label-free quantification study conducted on the serum samples from 10 healthy volunteers, 10 patients at early stages and 10 patients at advanced stages of lung adenocarcinoma, aiming to identify serum proteins that can be used as lung cancer biomarkers. The workflow was divided into four sections: 1) serum sample preparations involving immunodepletion of 14 high-abundance proteins, enzymatic deglycosylation in H218O, trypsin digestion and nano-reversed phase liquid chromatography; 2) acquisition of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) data from 30 cases; 3) label-free quantification and statistical analysis using Genedata Expressionist® platform; and 4) candidate biomarker protein identification by retrospective MS/MS acquisition. Deglycosylation was the critical step in the sample preparation phase as it could reduce spectral complexity caused by the presence of glycopeptides with heterogeneous glycan structures. Here we utilized H218O to incorporate 18O into aspartic acid residues that occurred during the deglycosylation reaction, and thus the deglycosylation sites resulted in the specific +3 Da mass shift allowing unambiguous determination of deglycosylated peptides and their glycosylation sites. We have shown here that 7.5 % of the identified peptides were N-glycosylated. This high level of glycopeptide occurrence, attributable to the fact that over 90 % of proteins in serum are glycoproteins, suggests that deglycosylation process would significantly simplify the peptide composition, which is critical for in-depth mass spectrometric quantification analyses of serum samples. As the result of label-free quantification analysis, approximately 15,000 peptide species (as peak clusters) were detected from 30 serum samples. 25 peptides were screened by one-way analysis of variance to be differentially represented among the three sample groups (normal, early and advanced lung cancer cases, p < 0.01). 16 peptides were specifically detected in normal serum and 9 showed increased levels in advanced stage cases, of which 2 were also detected at higher levels in early-stage cases than normal sera (p < 0.05). These peptides were subjected to MS/MS acquisition, referring back to the original data to select maximally detectable spot on the archived MALDI target plate for increased chance of identification. In conclusion, the combination of H218O deglycosylation, MALDI MS and label-free quantification can work effectively for precise quantification of glycoprotein-rich clinical specimens, allowing for the screening of serum biomarkers for cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4569.

Liquid Chromatography-Tandem and MALDI Imaging Mass Spectrometry Analyses of RCL2/CS100-Fixed, Paraffin-Embedded Tissues: Proteomics Evaluation of an Alternate Fixative for Biomarker Discovery

Human tissues are an important source of biological material for the discovery of novel biomarkers. Fresh-frozen tissue could represent an ideal supply of archival material for molecular investigations. However, immediate flash freezing is usually not possible, especially for rare or valuable tissue samples such as biopsies. Here, we investigated the compatibility of RCL2/CS100, a non-cross-linking, nontoxic, and nonvolatile organic fixative, with shotgun proteomic analyses. Several protein extraction protocols compatible with mass spectrometry were investigated from RCL2/CS100-fixed and fresh-frozen colonic mucosa, breast, and prostate tissues. The peptides and proteins identified from RCL2/CS100 tissue were then comprehensively compared with those identified from matched fresh-frozen tissues using a bottom-up strategy based on nano-reversed phase liquid chromatography coupled with tandem mass spectrometry (nanoRPLC-MS/MS). Results showed that similar peptides could be identified in both archival conditions and the proteome coverage was not obviously compromised by the RCL2/CS100 fixation process. NanoRPLC-MS/MS of laser capture microdissected RCL2/CS100-fixed tissues gave the same amount of biological information as that recovered from whole RCL2/CS100-fixed or frozen tissues. We next performed MALDI tissue profiling and imaging mass spectrometry and observed a high level of agreement in protein expression as well as excellent agreement between the images obtained from RCL2/CS100-fixed and fresh-frozen tissue samples. These results suggest that RCL2/CS100-fixed tissues are suitable for shotgun proteomic analyses and tissue imaging. More importantly, this alternate fixative opens the door to the analysis of small, valuable, and rare target lesions that are usually inaccessible to complementary biomarker-driven genomic and proteomic research.

Quantitative Profiling of Polar Cationic Metabolites in Human Cerebrospinal Fluid by Reversed-Phase Nanoliquid Chromatography/Mass Spectrometry

Reversed-phase (RP) nanoliquid chromatography (LC)/mass spectrometry (MS) is widely used for proteome analysis, but hydrophilic metabolites are poorly retained on RP columns. We describe here the development and application of an efficient, robust, and quantitative nano-LC/MS method for cationic metabolome analysis in the positive ionization mode without any derivatization of analytes. Various stationary phases for nano-LC, coating of the internal wall of the capillary column, and various mobile phases were evaluated in terms of separation and peak shapes for 33 hydrophilic metabolites, including nonderivatized amino acids. Polar cationic compounds were strongly bound to mixed-functional RP with cation exchange mode resin, and the best separation was obtained with hydrophilic internal wall coating and a two-step trifluoroacetic acid (TFA) gradient in methanol as the mobile phase. Simple, but optimized, sample processing and the use of a high content of methanol allowed robust nano-LC/MS analysis. Our developed method was applied for biomarker discovery in Alzheimer's disease (AD). Several hundred peaks were detected from 10 microL of cerebrospinal fluid (CSF). In a principal component analysis (PCA) plot using peak intensities without normalization, peak separation depended on the experimental date, not disease state. Therefore, constant amounts of two stable isotope-labeled amino acids, Val and Lys, were added as internal standards (ISs) to each sample before processing. These ISs were eluted in different gradient slopes in the two-step gradient, and the normalized peak ratios using the corresponding ISs gave a unique group of PCA scores which could distinguish AD CSF samples from age-matched control CSF samples.

The Influence of Sample Preparation and Replicate Analyses on HeLa Cell Phosphoproteome Coverage

Ongoing optimization of proteomic methodologies seeks to improve both the coverage and confidence of protein identifications. The optimization of sample preparation, inclusion of technical replicates (repeated instrumental analysis of the same sample), and biological replicates (multiple individual samples) are crucial in proteomic studies to avoid the pitfalls associated with single point analysis and under-sampling. Phosphopeptides were isolated from HeLa cells and analyzed by nano-reversed phase liquid chromatography electrospray ionization tandem mass spectrometry (nano-RP-LC-MS/MS). We observed that a detergent-based protein extraction approach, followed with additional steps for nucleic acid removal, provided a simple alternative to the broadly used Trizol extraction. The evaluation of four technical replicates demonstrated measurement reproducibility with low percent variance in peptide responses at approximately 3%, where additional peptide identifications were made with each added technical replicate. The inclusion of six technical replicates for moderately complex protein extracts (approximately 4000 uniquely identified peptides per data set) affords the optimal collection of peptide information.

Proteomic Analysis of Human Follicular Fluid Using an Alternative Bottom-Up Approach

Human follicular fluid (hFF) is the in vivo environment of oocytes during follicular maturation in the ovaries. It contains a huge variety of compounds such as, e.g., proteins that might play an important role in follicular development and oocyte growth. Previous proteomic studies on follicular fluid have isolated and already identified a certain number of proteins. Nevertheless, only a small part of proteins present in follicular fluid have been covered so far and a large number have still not been identified. Therefore, the need for new, more resolving, and sensitive approaches in proteome research is evident. We utilized a proteomic setup based on in solution isoelectric focusing (IEF) and reversed-phase nanoliquid chromatography coupled to matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (nano-LC MALDI TOF/TOF MS) for in depth protein analysis of human follicular fluid samples of patients undergoing controlled ovarian hyper stimulation (COH) for in vitro fertilization therapy (IVF). This approach led to the significant identification of 69 proteins, where 32 have not been reported before to be found in human follicular fluid with proteomic methods. Among these findings, at least two relevant compounds essentially involved in hormone secretion regulation during the folliculogenetic process were identified: sex hormone binding globulin (SHBG) and inhibin A (INHA). To confirm these results, both proteins were further validated by immunoassays.

Tissue proteomics using capillary isoelectric focusing-based multidimensional separations

The capabilities of capillary isoelectric focusing-based multidimensional separations for performing proteome analysis from minute samples create new opportunities in the pursuit of biomarker discovery using enriched and selected cell populations procured from tissue specimens. In this article, recent advances in online integration of capillary isoelectric focusing with nano-reversed phase liquid chromatography for achieving high-resolution peptide and protein separations prior to mass spectrometry analysis are reviewed, along with its potential application to tissue proteomics. These proteome technological advances combined with recently developed tissue microdissection techniques, provide powerful tools for those seeking to gain a greater understanding at the global level of the cellular machinery associated with human diseases such as cancer.

Effects of chromatography conditions on intact protein separations for top-down proteomics

For top-down proteomics, nano-reversed phase liquid chromatography (RPLC) plays a major role in both single and multidimensional protein separations in an effort to increase the overall peak capacity for the resolution of complex protein mixtures prior to mass spectrometry analysis. Effects of various chromatography conditions, including alkyl chain length in the stationary phase, capillary column temperature, and ion-pairing agent, on the resolution of intact proteins are studied using nano-RPLC-electrospray ionization-mass spectrometry. Optimal chromatography conditions include the use of C 18 column heated at 60 °C and the addition of trifluoroacetic acid instead of heptafluorobutyric acid as the ion-paring agent in the mobile phase. Under optimized chromatography conditions, there are no significant differences in the separation performance of yeast cell lysates present in the native versus denatured states. Denatured yeast proteins resolved and eluted from nano-RPLC can be subjected to proteolytic digestion in an on- or off-line approach to provide improved protein sequence coverage toward protein identification in a combined top-down/bottom-up proteome platform.

Integrated Capillary Isoelectric Focusing/Nano-reversed Phase Liquid Chromatography Coupled with ESI−MS for Characterization of Intact Yeast Proteins

An integrated protein concentration/separation platform, combining capillary isoelectric focusing (CIEF) with nano-reversed phase liquid chromatography (nano-RPLC), is developed to provide significant protein concentration and high resolving power for the analysis of complex protein mixtures. Upon completion of protein focusing, the proteins are sequentially and hydrodynamically loaded into individual trap columns using a group of microinjection and microselection valves. Repeated pro-tein loadings and injections into trap columns are carried out automatically until the entire CIEF cap-illary content is sampled and fractionated. Each CIEF fraction "parked" in separate trap columns is further resolved using nano-RPLC, and the eluants are analyzed using electrospray ionization-mass spectrometry.

Discovery of ovarian cancer biomarkers in serum using NanoLC electrospray ionization TOF and FT-ICR mass spectrometry.

Treatment of cancer patients is greatly facilitated by detection of the cancer prior to metastasis. One of the obstacles to early cancer detection is the lack of availability of biomarkers with sufficient specificity. With modern differential proteomic techniques, the potential exists to identify high specificity cancer biomarkers. We have delineated a set of protocols for the isolation and identification of serum biomarkers for ovarian cancer that exist in the low molecular weight serum fraction. After isolation of the low molecular weight fraction by ultrafiltration, the potential biomarkers are separated by reversed phase nano liquid chromatography. Detection via TOF or FT-ICR yields a data set for each sample. We compared stage III/IV ovarian cancer serum with postmenopausal age-matched controls. Using bioinformatics tools developed at Mayo, we normalized each sample for intensity and chromatographic alignment. Normalized data sets are subsequently compared and potential biomarkers identified. Several candidate biomarkers were found. One of these contains the sequence of fibrinopeptide-A known to be elevated in many types of cancer including ovarian cancer. The protocols utilized will be examined and would be applicable to a wide variety of cancers or disease states.