Proteome profile of human breast cancer tissue generated by LC-ESI-MS/MS combined with sequential protein precipitation and solubilization

Comprehensive proteome profiling of breast cancer tissue samples is challenging, as the tissue samples contain many proteins with varying concentrations and modifications. We report an effective sample preparation strategy combined with liquid chromatography (LC) electrospray ionization (ESI) quadrupole time-of-flight (QTOF) MS/MS for proteome analysis of human breast cancer tissue. The complexity of the breast cancer tissue proteome was reduced by using protein precipitation from a tissue extract, followed by sequential protein solubilization in solvents of different solubilizing strength. The individual fractions of protein mixtures or subproteomes were subjected to trypsin digestion and the resultant peptides were separated by strong cation exchange (SCX) chromatography, followed by reversed-phase capillary LC combined with high resolution and high accuracy ESI-QTOF MS/MS. This approach identified 14407 unique peptides from 3749 different proteins based on peptide matches with scores above the threshold scores at the 95% confidence level in MASCOT database search of the acquired MS/MS spectra. The false positive rate of peptide matches was determined to be 0.95% by using the target-decoy sequence search strategy. On the basis of gene ontology categorization, the identified proteins represented a wide variety of biological functions, cellular processes, and cellular locations.     

Differential expression analysis of Escherichia coli proteins using a novel software for relative quantitation of LC-MS/MS data

The study of changes in protein levels between samples derived from cells representing different biological conditions is a key to the understanding of cellular function. There are two main methods available that allow both for global scanning for significantly varying proteins and targeted profiling of proteins of interest. One method is based on 2-D gel electrophoresis and image analysis of labelled proteins. The other method is based on LC-MS/MS analysis of either unlabelled peptides or peptides derived from isotopically labelled proteins or peptides. In this study, the non-labelling approach was used involving a new software, DeCyder MS Differential Analysis Software (DeCyder MS) intended for automated detection and relative quantitation of unlabelled peptides in LC-MS/MS data.Total protein extracts of E. coli strains expressing varying levels of dihydrofolate reductase and integron integrase were digested with trypsin and analyzed using a nanoscale liquid chromatography system, Ettan MDLC, online connected to an LTQTM linear ion-trap mass spectrometer fitted with a nanospray interface. Acquired MS data were subjected to DeCyder MS analysis where 2-D representations of the peptide patterns from individual LC-MS/MS analyses were matched and compared.This approach to unlabelled quantitative analysis of the E. coli proteome resulted in relative protein abundances that were in good agreement with results obtained from traditional methods for measuring protein levels.     

Characterization of individual mouse cerebrospinal fluid proteomes

Analysis of cerebrospinal fluid (CSF) offers key insight into the status of the CNS. Characterization of murine CSF proteomes can provide a valuable resource for studying CNS injury and disease in animal models. However, the small volume of CSF in mice has thus far limited individual mouse proteome characterization. Through nonterminal CSF extractions in C57Bl/6 mice and high‐resolution 2D‐LC MS/MS analysis of individual murine samples, we report the most comprehensive proteome characterization of individual murine CSF to date. We identified a total of 566 unique proteins, including 128 proteins from three individual CSF samples that have been previously identified in brain tissue. Our methods and analysis provide a mechanism for individual murine CSF proteome analysis. The data are available in the ProteomeXchange with identifier PXD000248 ( http://proteomecentral.proteomexchange.org/dataset/PXD000248 ).     

A capillary liquid chromatographic/tandem mass spectrometric method for the quantification of gamma-aminobutyric acid in human plasma and cerebrospinal fluid

A sensitive and reliable method for the determination of gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter, in human plasma and cerebrospinal fluid (CSF) has been developed. The method is based on capillary liquid chromatography (LC)/tandem mass spectrometry (MS/MS) using deuterium-labeled GABA (gamma-aminobutyric acid-2,2-D(2), GABA-d(2)) as internal standard. Pre-column derivatization with 7-fluoro-4-nitrobenzoxadiazole (NBD-F) was deployed, allowing both effective in-line pre-concentration and sensitive tandem MS detection of the analyte. An extraction column (10 mm x 0.25 mm, 7 microm, C(18)) was used for preconcentrating and stacking the sample. Separation was carried out on an analytical column (50 mm x 0.25 mm, 5 microm, C(18)). Characteristic precursor-to-product ion transitions, m/z 267--> 249 (for NBD-GABA) and m/z 269--> 251 (for NBD-GABA-d(2)) were monitored for the quantification. A linear calibration curve from 10 to 250 ng/mL GABA with an r(2) value of 0.9994 was obtained. Detection limit was estimated to be 5.00 ng/mL GABA (S/N = 3). Human plasma and CSF samples were analyzed. The concentrations of GABA were found to be 98.6 +/- 33.9 ng/mL (mean +/- S.D., n = 12), and 44.3 +/- 10.0 ng/mL (n = 6) in plasma and CSF, respectively.     

Proteomic profiling of human stem cells derived from umbilical cord blood

CD34+ preparations from five different umbilical cord samples were compared with respect to their proteome profile using 2-D gel electrophoresis. Fifty-two protein spots were found to match in all preparations referring to the high heterogeneity of such samples indicating a not fully developed (or instable) proteome of stem cells. All matching spots were subjected to in-gel digestion and nano-LC-MS/MS sequence analysis, from which 22 proteins were unambiguously identified.     

Inhibition of intrinsic proteolytic activities moderates preanalytical variability and instability of human plasma

Human plasma and serum proteins are subject to intrinsic proteolytic degradation both during and after blood collection. By monitoring peptides, we investigated the stability of plasma and serum samples and the effects of anticoagulants and protease inhibitors on the plasma samples. Serum and plasma were subjected to time-course incubation, and the peptides (750-3200 Da) were extracted and analyzed with MALDI-TOF MS. Peptides of interest were further identified by MALDI-TOF/TOF MS and ESI-MS/MS analyses. Our observations indicate that plasma peptides are significantly different from serum peptides. Intrinsic proteases cause these differences between plasma and serum samples, as well as the differences among three plasma samples using either EDTA, sodium citrate, or heparin as the anticoagulant, which accounts for partial inhibitory effects on plasma proteolytic activities. Proteases and peptidases, including both aminopeptidases and carboxypeptidases, also cause time-dependent, sequential generation and digestion of the peptides in serum and all three plasmas, specifically during early sample collection and processing. Protease inhibitors within an EDTA-plasma-collection device inhibit both intrinsic plasma peptidases and proteases and moderate the time-dependent changes of the plasma peptides, including bradykinin, and complement C4- and C3- derived peptides. Our results suggest that mixing protease inhibitors immediately with blood during blood collection provides enhanced stabilization of the plasma proteome.     

Integrated analysis of the cerebrospinal fluid peptidome and proteome

Cerebrospinal fluid (CSF) is the only body fluid in direct contact with the brain and thus is a potential source of biomarkers. Furthermore, CSF serves as a medium of endocrine signaling and contains a multitude of regulatory peptides. A combined study of the peptidome and proteome of CSF or any other body fluid has not been reported previously. We report confident identification in CSF of 563 peptide products derived from 91 precursor proteins as well as a high confidence CSF proteome of 798 proteins. For the CSF peptidome, we use high accuracy mass spectrometry (MS) for MS and MS/MS modes, allowing unambiguous identification of neuropeptides. Combination of the peptidome and proteome data suggests that enzymatic processing of membrane proteins causes release of their extracellular parts into CSF. The CSF proteome has only partial overlap with the plasma proteome, thus it is produced locally rather than deriving from plasma. Our work offers insights into CSF composition and origin.     

A proteomic study of the HUPO Plasma Proteome Project's pilot samples using an accurate mass and time tag strategy

Characterization of the human blood plasma proteome is critical to the discovery of routinely useful clinical biomarkers. We used an accurate mass and time (AMT) tag strategy with high-resolution mass accuracy cLC-FT-ICR MS to perform a global proteomic analysis of pilot study samples as part of the HUPO Plasma Proteome Project. HUPO reference serum and citrated plasma samples from African Americans, Asian Americans, and Caucasian Americans were analyzed, in addition to a Pacific Northwest National Laboratory reference serum and plasma. The AMT tag strategy allowed us to leverage two previously published "shotgun" proteomics experiments to perform global analyses on these samples in triplicate in less than 4 days total analysis time. A total of 722 (22% with multiple peptide identifications) International Protein Index redundant proteins, or 377 protein families by ProteinProphet, were identified over the six individual HUPO serum and plasma samples. The samples yielded a similar number of identified redundant proteins in the plasma samples (average 446 +/- 23) as found in the serum samples (average 440 +/- 20). These proteins were identified by an average of 956 +/- 35 unique peptides in plasma and 930 +/- 11 unique peptides in serum. In addition to this high-throughput analysis, the AMT tag approach was used with a Z-score normalization to compare relative protein abundances. This analysis highlighted both known differences in serum and citrated plasma such as fibrinogens, and reproducible differences in peptide abundances from proteins such as soluble activin receptor-like kinase 7b and glycoprotein m6b. The AMT tag strategy not only improved our sample throughput but also provided a basis for estimated quantitation.     

Optimising ovine cerebrospinal fluid preparation for two-dimensional gel electrophoresis

Biomarkers for neurodegenerative disorders are potentially present in cerebrospinal fluid (CSF) and can be detected using proteomic technologies. Since CSF is high in salt and low in protein, its study by proteomic methods requires appropriate sample preparation. In this study, we applied four different sample treatments to the same ovine CSF sample. Precipitation with acetone or using a 2-D Clean-Up Kit (GE Healthcare BioSciences, Little Chalfont, UK) preserved more proteins, and produced more gel spots than spin columns from Sigma and Bio-Rad. A 53-kDa spot, identified by MS/MS as transthyretin (TTR) tetramer, was not detected in samples treated with the 2-D Clean-Up Kit, though it was always present on all gels prepared using the other three methods. Western immunoblotting confirmed the low recovery of tetrameric TTR by the 2-D Clean-Up Kit and showed that the tetrameric form of TTR predominated in ovine but not in rat CSF. In one ovine CSF sample haemoglobin was found, indicating blood contamination. We conclude that acetone precipitation is a simple and efficient way to prepare ovine CSF for 2-DE. The use of the 2-D Clean-Up Kit leads to the disappearance of tetrameric TTR only from ovine CSF proteome.     

Different patterns of central and peripheral beta EP, beta LPH and ACTH throughout life

The aim of the present study was to evaluate the relationship between central and peripheral concentrations of proopiocortin-related peptides in different periods of life. One hundred and eighty-nine plasma samples from normal subjects (18-87 years) obtained in basal conditions, and 20 cerebrospinal fluid (CSF) samples obtained by lumbar puncture from healthy volunteers (18-75 years) were studied. beta Lipotropin (beta LPH), beta endorphin (beta EP) and ACTH were measured by specific RIA after silicic acid plasma extraction and gel chromatography (beta LPH and beta EP). No sex differences were found in the patterns of the three peptides either in the plasma or in CSF. In the plasma samples, both beta LPH and beta EP concentrations showed a pattern throughout life which was expressed by a paraboloid function with the lowest values found in young and old subjects and with peaks at 51.3 and 48.2 years, respectively. On the contrary, ACTH values failed to be represented by a significant linear or curvilinear regression and presented only a slight decrease in subjects over 75 years of age. CSF levels of beta LPH were significantly lower in 45-76 year old subjects (18.8 +/- 12.6 fmol/ml, M +/- SD) than in 18-44 year old subjects (34.5 +/- 15.8; p less than 0.05), as were those of beta EP (elderly: 41.2 +/- 19.7; young: 94.2 +/- 36.7; p less than 0.05), which showed a significantly linear inverse correlation with age (r = 0.6062, p less than 0.01). These CSF samples did not show any ACTH variations connected with age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the HUPO Brain Proteome reference samples using 2-D DIGE and 2-D LC-MS/MS

Within the Human Proteome Organization (HUPO) Brain Proteome Project, a pilot study was launched with reference samples shipped to nine international laboratories (see Hamacher et al., this Special Issue) to evaluate different proteome approaches in neuroscience and to build up a first version of a brain protein database. One part of the study addresses quantitative proteome alterations between three developmental stages (embryonic day 16; postnatal day 7; 8 weeks) of mouse brains. Five brains per stage were differentially analyzed by 2-D DIGE using internal standardization and overlapping pH gradients (pH 4-7 and 6-9). In total, 214 protein spots showing stage-dependent intensity alterations (> two-fold) were detected, 56 of which were identified. Several of them, e.g. members of the dihydropyrimidinase family, are known to be associated with brain development. To feed the HUPO BPP brain protein database, a robust 2-D LC-MS/MS method was applied to murine postnatal day 7 and human post-mortem brain samples. Using MASCOT and the IPI database, 350 human and 481 mouse proteins could be identified by at least two different peptides. The data are accessible through the PRIDE database (http://www.ebi.ac.uk/pride/).     

Quantitative proteome analysis of human plasma following in vivo lipopolysaccharide administration using 16O/18O labeling and the accurate mass and time tag approach

Identification of novel diagnostic or therapeutic biomarkers from human blood plasma would benefit significantly from quantitative measurements of the proteome constituents over a range of physiological conditions. Herein we describe an initial demonstration of proteome-wide quantitative analysis of human plasma. The approach utilizes postdigestion trypsin-catalyzed 16O/18O peptide labeling, two-dimensional LC-FTICR mass spectrometry, and the accurate mass and time (AMT) tag strategy to identify and quantify peptides/proteins from complex samples. A peptide accurate mass and LC elution time AMT tag data base was initially generated using MS/MS following extensive multidimensional LC separations to provide the basis for subsequent peptide identifications. The AMT tag data base contains >8,000 putative identified peptides, providing 938 confident plasma protein identifications. The quantitative approach was applied without depletion of high abundance proteins for comparative analyses of plasma samples from an individual prior to and 9 h after lipopolysaccharide (LPS) administration. Accurate quantification of changes in protein abundance was demonstrated by both 1:1 labeling of control plasma and the comparison between the plasma samples following LPS administration. A total of 429 distinct plasma proteins were quantified from the comparative analyses, and the protein abundances for 25 proteins, including several known inflammatory response mediators, were observed to change significantly following LPS administration.     

Establishing the Proteome of Normal Human Cerebrospinal Fluid

Background

Knowledge of the entire protein content, the proteome, of normal human cerebrospinal fluid (CSF) would enable insights into neurologic and psychiatric disorders. Until now technologic hurdles and access to true normal samples hindered attaining this goal.

Methods and Principal Findings

We applied immunoaffinity separation and high sensitivity and resolution liquid chromatography-mass spectrometry to examine CSF from healthy normal individuals. 2630 proteins in CSF from normal subjects were identified, of which 56% were CSF-specific, not found in the much larger set of 3654 proteins we have identified in plasma. We also examined CSF from groups of subjects previously examined by others as surrogates for normals where neurologic symptoms warranted a lumbar puncture but where clinical laboratory were reported as normal. We found statistically significant differences between their CSF proteins and our non-neurological normals. We also examined CSF from 10 volunteer subjects who had lumbar punctures at least 4 weeks apart and found that there was little variability in CSF proteins in an individual as compared to subject to subject.

Conclusions

Our results represent the most comprehensive characterization of true normal CSF to date. This normal CSF proteome establishes a comparative standard and basis for investigations into a variety of diseases with neurological and psychiatric features.     

Utility of electrophoretically derived protein mass estimates as additional constraints in proteome analysis of human serum based on MS/MS analysis

The proteome of a HUPO human serum reference sample was analyzed using multidimensional separation techniques at both the protein and the peptide levels. To eliminate false-positive identifications from the search results, we employed a data filtering method using molecular weight (MW) correlations derived from denaturing 1-DE. First, the six most abundant serum proteins were removed from the sample using immunoaffinity chromatography. 1-DE was then used to fractionate the remaining serum proteins according to the MW. Gel bands were isolated and in-gel digested with trypsin, and the resulting peptides were analyzed by 2-D LC/ESI-MS/MS. A SEQUEST search using the MS/MS results identified 494 proteins. Of these, 202 were excluded formally using protein data filtering as they were single-assignment proteins and their theoretical and electrophoretically-derived MWs did not correlate at high confidence. To evaluate this method, the results were compared with those of 1-D LC/MALDI-TOF/TOF and HUPO Plasma Proteome Project analyses. Our data filtering approach proved valuable in analysis of complex, large-scale proteomes such as human serum.     

Clinical-scale high-throughput human plasma proteome analysis: lung adenocarcinoma

Clinical proteomics requires the stable and reproducible analysis of a large number of human samples. We report a high-throughput comprehensive protein profiling system comprising a fully automated, on-line, two-dimensional microflow liquid chromatography/tandem mass spectrometry (2-D microLC-MS/MS) system for use in clinical proteomics. A linear ion-trap mass spectrometer (ITMS) also known as a 2-D ITMS instrument, which is characterized by high scan speed, was incorporated into the microLC-MS/MS system in order to obtain highly improved sensitivity and resolution in MS/MS acquisition. This system was used to evaluate bovine serum albumin and human 26S proteasome. Application of these high-throughput microLC conditions and the 2-D ITMS resulted in a 10-fold increase in sensitivity in protein identification. Additionally, peptide fragments from the 26S proteasome were identified three-fold more efficiently than by the conventional 3-D ITMS instrument. In this study, the 2-D microLC-MS/MS system that uses linear 2-D ITMS has been applied for the plasma proteome analysis of a few samples from healthy individuals and lung adenocarcinoma patients. Using the 2-D and 1-D microLC-MS/MS analyses, approximately 250 and 100 different proteins were detected, respectively, in each HSA- and IgG-depleted sample, which corresponds to only 0.4 microL of blood plasma. Automatic operation enabled the completion of a single run of the entire 1-D and 2-D microLC-MS/MS analyses within 11 h. Investigation of the data extracted from the protein identification datasets of both healthy and adenocarcinoma groups revealed that several of the group-specific proteins could be candidate protein disease markers expressed in the human blood plasma. Consequently, it was demonstrated that this high-throughput microLC-MS/MS protein profiling system would be practically applicable to the discovery of protein disease markers, which is the primary objective in clinical plasma proteome projects.     

Preliminary 2-D chromatographic investigation of the human stem cell proteome

Stem cells represent a promising tool for the treatment of various hematopoietic diseases. In order to identify stem cell-specific proteins, the proteome of human stem cells from umbilical cord blood was explored for the first time. For this purpose, the crude lysate of 4 x 10(5) CD34+ cells was subjected to in solution trypsin digestion. The resulting peptides were then separated via cation exchange followed by reversed phase chromatography and analyzed by nanospray MS/MS. Database search revealed a total of 215 proteins which could be reliably identified. To obtain a more complete picture of the human stell cell proteome and to also access low abundant proteins, pooling of more than one CD34+ preparations seems necessary in order to increase the cell number and thus the protein content.     

High abundance protein profiling of cystic fibrosis lung epithelial cells

Protein profiles of cultured cystic fibrosis (CF) lung epithelial cells were analyzed by two-dimensional gel electrophoresis and mass spectrometry (MS). The analysis gave rise to a protein map over the pI range of 4-7, and a molecular weight range of ca. 100-10 kDa. The map contains 194 identified proteins, which were detectable by silver stain. All silver stained features were identified by matrix-assisted laser desorption/ionization-time of flight MS of tryptic peptides. Some proteins were found to be represented by multiple features on the 2-D gel. Among the high abundance proteins identified were sets of proteins associated with inflammation, including the classical NFkappaB, p65 (RelA) and NFkappaB, p65 (RelB). We suggest that this composite atlas of the high abundance CF lung epithelial proteome will serve as a reference database for future studies of candidate CF drugs, validating different approaches to CFTR gene therapy, and analogous investigations of other types of human lung disorders.     

Assessment of protein stability in cerebrospinal fluid using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry protein profiling

Recent studies have evaluated proper acquisition and storage procedures for the use of serum or plasma for mass spectrometry (MS)-based proteomics. The present study examines the proteome stability of human cerebrospinal fluid (CSF) over time at 23°C (room temperature) and 4°C using surface-enhanced laser desorption/ionization time-of-flight MS. Data analysis revealed that statistically significant differences in protein profiles are apparent within 4 h at 23°C and between 6 and 8 h at 4°C. Inclusion of protease and phosphatase inhibitor cocktails into the CSF samples failed to significantly reduce proteome alterations over time. We conclude that MS-based proteomic analysis of CSF requires careful assessment of sample collection procedures for rapid and optimal sample acquisition and storage.     

The plasma peptidome

Background

It may be possible to discover new diagnostic or therapeutic peptides or proteins from blood plasma using LC–ESI–MS/MS to identify, with a linear quadrupole ion trap to identify, quantify and compare the statistical distributions of peptides cleaved ex vivo from plasma samples from different clinical populations.

Methods

A systematic method for the organic fractionation of plasma peptides was applied to identify and quantify the endogenous tryptic peptides from human plasma from multiple institutions by C18 HPLC followed nano electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) with a linear quadrupole ion trap. The endogenous tryptic peptides, or tryptic phospho peptides (i.e. without exogenous digestion), were extracted in a mixture of organic solvent and water, dried and collected by preparative C18. The tryptic peptides from 6 institutions with 12 different disease and normal EDTA plasma populations, alongside ice cold controls for pre-analytical variation, were characterized by mass spectrometry. Each patient plasma was precipitated in 90% acetonitrile and the endogenous tryptic peptides extracted by a stepwise gradient of increasing water and then formic acid resulting in 10 sub-fractions. The fractionated peptides were manually collected over preparative C18 and injected for 1508 LC–ESI–MS/MS experiments analyzed in SQL Server R.

Results

Peptides that were cleaved in human plasma by a tryptic activity ex vivo provided convenient and sensitive access to most human proteins in plasma that show differences in the frequency or intensity of proteins observed across populations that may have clinical significance. Combination of step wise organic extraction of 200 μL of plasma with nano electrospray resulted in the confident identification and quantification ~?14,000 gene symbols by X!TANDEM that is the largest number of blood proteins identified to date and shows that you can monitor the ex vivo proteolysis of most human proteins, including interleukins, from blood. A total of 15,968,550 MS/MS spectra ≥?E4 intensity counts were correlated by the SEQUEST and X!TANDEM algorithms to a federated library of 157,478 protein sequences that were filtered for best charge state (2+ or 3+) and peptide sequence in SQL Server resulting in 1,916,672 distinct best-fit peptide correlations for analysis with the R statistical system. SEQUEST identified some 140,054 protein accessions, or some ~?26,000 gene symbols, proteins or loci, with at least 5 independent correlations. The X!TANDEM algorithm made at least 5 best fit correlations to more than 14,000 protein gene symbols with p-values and FDR corrected q-values of ~?0.001 or less. Log₁₀ peptide intensity values showed a Gaussian distribution from E8 to E4 arbitrary counts by quantile plot, and significant variation in average precursor intensity across the disease and controls treatments by ANOVA with means compared by the Tukey–Kramer test. STRING analysis of the top 2000 gene symbols showed a tight association of cellular proteins that were apparently present in the plasma as protein complexes with related cellular components, molecular functions and biological processes.

Conclusions

The random and independent sampling of pre-fractionated blood peptides by LC-ESI-MS/MS with SQL Server-R analysis revealed the largest plasma proteome to date and was a practical method to quantify and compare the frequency or log₁₀ intensity of individual proteins cleaved ex vivo across populations of plasma samples from multiple clinical locations to discover treatment-specific variation using classical statistics suitable for clinical science. It was possible to identify and quantify nearly all human proteins from EDTA plasma and compare the results of thousands of LC–ESI–MS/MS experiments from multiple clinical populations using standard database methods in SQL Server and classical statistical strategies in the R data analysis system.