Investigation of alkaline effects on Protein A affinity ligands and resins using high resolution mass spectrometry.

In this study, the enhanced alkaline stability of Protein A ligands and resins designed by protein engineering approaches is demonstrated. High throughput PreDictor™ plates were used to evaluate and compare the human Immunoglobulin G (IgG) static binding capacities (SBC) of MabSelect SuRe™ and MabSelect™ PrismA affinity chromatography (AC) resins after continuous incubation in 0.1-2.0 M NaOH for 1-72 h. The alkaline effect on the Protein A affinity ligand was studied by high resolution mass spectrometry (MS). The IgG binding capacity of the investigated AC resins show expected declining trends with increasing NaOH concentrations and incubation times. The decrease is larger for MabSelect SuRe than for MabSelect PrismA and occur at lower NaOH concentrations. MabSelect SuRe display high remaining binding capacity even after 72 h continuous incubation in 0.1 M NaOH, while higher concentrations induce an accentuated decline with incubation time. The MabSelect PrismA resin shows almost no effect on the binding capacity even after 72 h incubation in 0.5 M NaOH. Decline in capacity is first observed after 48 h incubation in 1.0 M NaOH, thus displaying the extreme alkaline stability of the PrismA affinity ligand. The MS analysis of the ligands, including a Protein A single B-domain, SuRe-domain and PrismA-domain clearly illustrate the increasing alkaline stability (B-domain < SuRe < PrismA) as the ligand has been refined using a protein engineering approach. Deamidation and ligand degradation could be monitored in relation to NaOH incubation conditions. Enzymatic digestion of MabSelect SuRe and MabSelect PrismA resins after alkaline incubation and LC-MS/MS peptide mapping facilitates identification and quantification of specific deamidation sites on the affinity ligand.

Removal of B. cereus cereulide toxin from monoclonal antibody bioprocess feed via two-step Protein A affinity and multimodal chromatography.

A rapid and sensitive liquid chromatography-mass spectrometry assay was developed and used to quantify emetic cereulide peptide exotoxin, which can be related to possible Bacillus cereus contamination in monoclonal antibody (mAb) bioprocess feeds. The assay limit of detection was 0.05 ng/mL (~1 fmol injected) and limit of quantification 0.16 ng/mL (~3 fmol injected) over a standard curve with >3 orders of magnitude linear dynamic range. The assay allowed quantification of toxin removal in an established two-step mAb purification process consisting of Protein A affinity chromatography followed by multi-modal anion exchange chromatography. Toxin content was ascertained in process stream sample fractions as well as on the Protein A affinity column. An optimized analytical method allowed separation of cereulide toxin from other mAb cell culture components within 6 min. Spiking experiments showed that samples should be collected in high (80% v/v) content acetonitrile to reduce nonspecific losses of the cereulide. The majority of mAb purification process-associated cereulide was detected in the Protein A flow through fraction, whereas only residual amounts were found in wash, strip, and elution fractions. Column cleaning-in-place (CIP) procedures were evaluated to prevent carryover between affinity capture cycles. No carryover was detected between cycles, however trace amounts of cereulide were extracted from the Protein A resin. Increasing the CIP NaOH concentration from 0.1 M to 0.5 M, and contact time from 15 min to 1 h, improved removal of residual cereulide from the resin. Applicability of CIP clearance of cereulide during Protein A chromatography was confirmed with three different mAb feeds. Post Protein A polishing, via target flow through on a multi-modal anion exchange chromatography column, resulted in a product pool with no detectable cereulide. Approximately 5 logs of reduction in cereulide concentration was obtained over the two-step chromatography process. Cereulide contamination is well known and of concern in food processing, however this research may be the first LC-MS quantification of cereulide contamination, and its clearance, in biopharmaceutical mAb processing. The analytical method may also be used to rapidly screen for cereulide contamination in upstream cell culture process streams, prior to downstream product purification. This will allow appropriate measures to be taken to reduce toxin exposure to downstream bioprocess raw materials, consumables and equipment.

De novo design and synthesis of a 30-cistron translation-factor module.

Two of the many goals of synthetic biology are synthesizing large biochemical systems and simplifying their assembly. While several genes have been assembled together by modular idempotent cloning, it is unclear if such simplified strategies scale to very large constructs for expression and purification of whole pathways. Here we synthesize from oligodeoxyribonucleotides a completely de-novo-designed, 58-kb multigene DNA. This BioBrick plasmid insert encodes 30 of the 31 translation factors of the PURE translation system, each His-tagged and in separate transcription cistrons. Dividing the insert between three high-copy expression plasmids enables the bulk purification of the aminoacyl-tRNA synthetases and translation factors necessary for affordable, scalable reconstitution of an in vitro transcription and translation system, PURE 3.0.

Increased Levels of Extracellular Microvesicle Markers and Decreased Levels of Endocytic/Exocytic Proteins in the Alzheimer's Disease Brain.

BACKGROUND: Alzheimer's disease (AD) is a chronic neurodegenerative disorder accounting for more than 50% of all dementia cases. AD neuropathology is characterized by the formation of extracellular plaques and intracellular neurofibrillary tangles consisting of aggregated amyloid-ß and tau, respectively. The disease mechanism has only been partially elucidated and is believed to also involve many other proteins. OBJECTIVE: This study intended to perform a proteomic profiling of post mortem AD brains and compare it with control brains as well as brains from other neurological diseases to gain insight into the disease pathology. METHODS: Here we used label-free shotgun mass spectrometry to analyze temporal neocortex samples from AD, other neurological disorders, and non-demented controls, in order to identify additional proteins that are altered in AD. The mass spectrometry results were verified by antibody suspension bead arrays. RESULTS: We found 50 proteins with altered levels between AD and control brains. The majority of these proteins were found at lower levels in AD. Pathway analyses revealed that several of the decreased proteins play a role in exocytic and endocytic pathways, whereas several of the increased proteins are related to extracellular vesicles. Using antibody-based analysis, we verified the mass spectrometry results for five representative proteins from this group of proteins (CD9, HSP72, PI42A, TALDO, and VAMP2) and GFAP, a marker for neuroinflammation. CONCLUSIONS: Several proteins involved in exo-endocytic pathways and extracellular vesicle functions display altered levels in the AD brain. We hypothesize that such changes may result in disturbed cellular clearance and a perturbed cell-to-cell communication that may contribute to neuronal dysfunction and cell death in AD.

Spinal Cord Stimulation Alters Protein Levels in the Cerebrospinal Fluid of Neuropathic Pain Patients: A Proteomic Mass Spectrometric Analysis.

OBJECTIVES: Electrical neuromodulation by spinal cord stimulation (SCS) is a well-established method for treatment of neuropathic pain. However, the mechanism behind the pain relieving effect in patients remains largely unknown. In this study, we target the human cerebrospinal fluid (CSF) proteome, a little investigated aspect of SCS mechanism of action. METHODS: Two different proteomic mass spectrometry protocols were used to analyze the CSF of 14 SCS responsive neuropathic pain patients. Each patient acted as his or her own control and protein content was compared when the stimulator was turned off for 48 hours, and after the stimulator had been used as normal for three weeks. RESULTS: Eighty-six proteins were statistically significantly altered in the CSF of neuropathic pain patients using SCS, when comparing the stimulator off condition to the stimulator on condition. The top 12 of the altered proteins are involved in neuroprotection (clusterin, gelsolin, mimecan, angiotensinogen, secretogranin-1, amyloid beta A4 protein), synaptic plasticity/learning/memory (gelsolin, apolipoprotein C1, apolipoprotein E, contactin-1, neural cell adhesion molecule L1-like protein), nociceptive signaling (neurosecretory protein VGF), and immune regulation (dickkopf-related protein 3). CONCLUSION: Previously unknown effects of SCS on levels of proteins involved in neuroprotection, nociceptive signaling, immune regulation, and synaptic plasticity are demonstrated. These findings, in the CSF of neuropathic pain patients, expand the picture of SCS effects on the neurochemical environment of the human spinal cord. An improved understanding of SCS mechanism may lead to new tracks of investigation and improved treatment strategies for neuropathic pain.

Quantification of 10 elements in human cerebrospinal fluid from chronic pain patients with and without spinal cord stimulation.

Neuropathic pain affects 1-10% of the general population and is caused by a lesion or disease of the somatosensory nervous system. Spinal cord stimulation (SCS), a method where implanted electrodes stimulate the spinal cord, has been successfully used to treat drug-resistant neuropathic pain, but the mechanism of action is largely unknown. Studies show that SCS changes the protein levels in CSF (cerebrospinal fluid) of pain patients. Several neurological conditions have been shown to alter the elemental composition of CSF. Therefore changes in the levels of ions and trace elements in the CSF may correspond to SCS use. This study used ICP-MS (Inductively coupled plasma mass spectrometry) and ICP-AES (Inductively coupled plasma atomic emission spectroscopy) to quantify 10 elements in CSF from chronic neuropathic pain patients using SCS. The element concentrations in CSF from patients with SCS treatment on/off, were measured. No effect on the element concentrations in CSF from treatment with SCS could be detected. Also, the elemental concentrations in pooled CSF from patients without chronic neuropathic pain was determined and compared to the patients using SCS. The concentration of the elements Ca, Sr, Na, K, P, Mg and Ti were, significantly higher in patients compared to the CSF-control.

Quantification of the brain proteome in Alzheimer's disease using multiplexed mass spectrometry.

We have compared the brain proteome in the temporal neocortex between Alzheimer's disease (AD) patients and non-AD individuals by using shotgun mass spectrometry based on a stable isotope dimethyl labeling. A total of 827 unique proteins were identified and quantitated. Of these, 227 proteins were found in at least 9 out of 10 AD/control pairs and were further subjected to statistical analysis. A total of 69 proteins showed different levels (p-value < 0.05) in AD versus control brain samples. Of these proteins, 37 were increased and 32 were decreased as compared to the non-AD subjects. Twenty-three proteins comprise novel proteins that have not previously been reported as related to AD, e.g., neuronal-specific septin-3, septin-2, septin-5, dihydropteridine reductase, and clathrin heavy chain 1. The proteins with altered levels in the AD brain represent a wide variety of pathways suggested to be involved in the disease pathogenesis, including energy metabolism, glycolysis, oxidative stress, apoptosis, signal transduction, and synaptic functioning. Apart from leading to new insights into the molecular mechanisms in AD, the findings provide us with possible novel candidates for future diagnostic and prognostic disease markers.

Identification of human cerebrospinal fluid proteins and their distribution in an in vitro microdialysis sampling system.

A qualitative study is presented on how proteins from a complex biological sample are distributed in a microdialysis sample system. A comparison between proteins identified in the human ventricular cerebrospinal fluid (CSF) sample, the collected dialysate and the proteins adsorbed onto the membrane was conducted. The microdialysis experiment was performed in vitro at 37°C for the duration of 24h. Thereafter, the membranes were removed from the catheter and the adsorbed proteins were tryptically digested using the on-surface enzymatic digestion (oSED) protocol. The CSF samples and the dialysates were digested using a standard in-solution trypsin digestion protocol. In the final phase, the samples were analysed using nano-liquid chromatography in combination with tandem mass spectrometry. In the four sample compartments analysed (CSF start, Membrane, Dialysate, CSF end) a total of 134 different proteins were found. However, most of the identified proteins (n=87) were uniquely found in one sample compartment only. Common CSF proteins such as albumin, apolipoproteins and cystatin C together with plasma proteins such as hemoglobin and fibrinogen were among the 11 proteins that were found in all samples. These proteins are present in high concentrations in CSF, which means that they effectively block out the detection signal of less abundant proteins. Therefore, only 25% of the proteins adsorbed onto the membrane were detected in the CSF compared with the dialysate that shared 44% of its proteins with the CSF. The proteins adsorbed onto the membrane were significantly more hydrophobic, had a lower instability index and more thermostable compared to the proteins in the CSF and the dialysate. The results suggest that proteins adsorbed onto the microdialysis membranes may escape detection because they are prevented from passing the membrane into the dialysate. Thus, the membrane needs to be examined after sample collection in order to better verify the protein content in the original sample. This is particularly important when searching for new protein biomarkers for neurodegenerative diseases.

Comparative study of label and label-free techniques using shotgun proteomics for relative protein quantification.

The analytical performance of three different strategies, iTRAQ (isobaric tag for relative and absolute quantification), dimethyl labeling (DML) and label free (LF) for relative protein quantification using shotgun proteomics have been evaluated. The methods have been explored using samples containing (i) Bovine proteins in known ratios and (ii) Bovine proteins in known ratios spiked into Escherichia coli. The latter case mimics the actual conditions in a typical biological sample with a few differentially expressed proteins and a bulk of proteins with unchanged ratios. Additionally, the evaluation was performed on both QStar and LTQ-FTICR mass spectrometers. LF LTQ-FTICR was found to have the highest proteome coverage while the highest accuracy based on the artificially regulated proteins was found for DML LTQ-FTICR (54%). A varying linearity (k: 0.55-1.16, r(2): 0.61-0.96) was shown for all methods within selected dynamic ranges. All methods were found to consistently underestimate Bovine protein ratios when matrix proteins were added. However, LF LTQ-FTICR was more tolerant toward a compression effect. A single peptide was demonstrated to be sufficient for a reliable quantification using iTRAQ. A ranking system utilizing several parameters important for quantitative proteomics demonstrated that the overall performance of the five different methods was; DML LTQ-FTICR>iTRAQ QStar>LF LTQ-FTICR>DML QStar>LF QStar.

Identification of injury specific proteins in a cell culture model of traumatic brain injury.

The complicated secondary molecular and cellular mechanisms following traumatic brain injury (TBI) are still not fully understood. In the present study, we have used mass spectrometry to identify injury specific proteins in an in vitro model of TBI. A standardized injury was induced by scalpel cuts through a mixed cell culture of astrocytes, oligodendrocytes and neurons. Twenty-four hours after the injury, cell culture medium and whole-cell fractions were collected for analysis. We found 53 medium proteins and 46 cell fraction proteins that were specifically expressed after injury and the known function of these proteins was elucidated by an extensive literature survey. By using time-lapse microscopy and immunostainings we could link a large proportion of the proteins to specific cellular processes that occur in response to trauma; including cell death, proliferation, lamellipodia formation, axonal regeneration, actin remodeling, migration and inflammation. A high percentage of the proteins uniquely expressed in the medium after injury were actin-related proteins, which normally are situated intracellularly. We show that two of these, ezrin and moesin, are expressed by astrocytes both in the cell culture model and in mouse brain subjected to experimental TBI. Interestingly, we found many inflammation-related proteins, despite the fact that cells were present in the culture. This study contributes with important knowledge about the cellular responses after trauma and identifies several potential cell-specific biomarkers.

Longitudinal characterization of the brain proteomes for the tg2576 amyloid mouse model using shotgun based mass spectrometry.

Neurodegenerative disorders are often defined pathologically by the presence of protein aggregates, such as amyloid plaques composed of ß-amyloid (Aß) peptide in Alzheimer's disease. Such aggregates are the result of abnormal protein accumulation and may lead to neuronal dysfunction and cell death. In this study, APPSWE transgenic mice (Tg2576), which overexpress the Swedish mutated form of human amyloid precursor protein (APP), were used to study the brain proteome associated with amyloid plaque deposition. The major aim of the study was to map and compare the Tg2576 model brain proteome profiles during pathology progression using a shotgun approach based on label free quantification with mass spectrometry. Overall, 1085 proteins were identified and longitudinally quantified. Principal component analysis (PCA) showed the appearance of the pathology onset between twelve and fifteen months, correlating with sharp amyloid plaque accumulation within the same ages. Cluster analysis followed by protein-protein interaction analysis revealed an age-dependent decrease in mitochondrial protein expression. We identified 57 significantly affected mitochondrial proteins, several of which have been reported to alter expression in neurological diseases. We also found ten proteins that are upregulated early in the amyloid driven pathology progression with high confidence, some of which are directly involved in the onset of mitochondrial apoptosis and may represent potential markers for use in human neurological diseases prognosis. Our results further contribute to identifying common pathological pathways involved in both aging and progressive neurodegenerative disorders enhancing the understanding of disease pathogenesis.

Comparison of extraction methods for the comprehensive analysis of mouse brain proteome using shotgun-based mass spectrometry.

This study compares 16 different extraction methods for the comprehensive extraction of mouse brain proteome in combination with "shotgun"-based mass spectrometry (MS). Membrane proteins (MPs) are responsible for a large part of the regulatory functions of the cell and are therefore of great interest to extract and analyze. Sixteen protein extraction protocols were evaluated in regards to protein yield and number of identified proteins with emphasis on MPs. The extracted proteins were delipidated, on-filter digested, and analyzed by reversed phase nanoliquid chromatography (RP-nanoLC) in combination with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using a 7 T hybrid LTQ-FT mass spectrometer. Detergent-based lysis buffers showed higher efficiencies and yields in the extraction of proteins from the brain tissue compared to solubilization with organic solvents or organic acids. The detergent octyl-ß-D-glucopyranoside gave the highest number of identified proteins (541) as well as numbers and percentages of identified MPs (29%). Detergent-based protocols are the best sample preparation tools for central nervous system (CNS) tissue and can readily be applied to screen for candidate biomarkers of neurological diseases.

Proteins in aqueous humor from cataract patients with and without pseudoexfoliation syndrome.

The aim of this study was to investigate the protein content in aqueous humor in eyes with and without pseudoexfoliations (PEX) and to evaluate the quantitative proteomics method, isobaric tagging for relative and absolute protein quantification (iTRAQ), in combination with two separation methods followed by matrix-assisted Laser desorption/ionization (MALDI) mass spectrometry and tandem mass spectrometry (MS/MS). During cataract surgery, samples of aqueous humor were collected from 20 eyes with PEX and from 18 control eyes. The relative concentrations of proteins in the pooled samples of ten PEX eyes and eight controls were evaluated after trypsin digestion and Labeling of the peptides with (iTRAQ) reagent. Two separation methods, Liquid chromatography (LC) and capillary electrophoresis (CE) were used, followed by MALDI mass spectrometry and MS/MS. Furthermore, 1D gel electrophoresis was performed on the remaining ten pooled PEX samples and ten control samples. The gel material was separated by nano-liquid chromatography (nano-LC) followed by Linear-ion-trap quadrupole Fourier transformation ion cyclotron resonance (FT-ICR). Fifty four proteins were identified in the LC runs and 24 with CE. The relative concentrations of beta-crystallines B2 and S were raised and those of angiotensinogen and osteopontin Lowered in the PEX sample compared to the control. The trends regarding beta-crystallines B2, angiotensinogen and osteopontin were confirmed by the 1D gel electrophoresis.

Multiplexed quantification of proteins adsorbed to surface-modified and non-modified microdialysis membranes.

A simple and straightforward method for discovery and quantification of proteins adsorbed onto delicate and sensitive membrane surfaces is presented. The adsorbed proteins were enzymatically cleaved while still adsorbed onto the membranes using an on-surface enzymatic digestion (oSED). This was followed by isobaric tagging, nanoliquid chromatography, and tandem mass spectrometry. Protein adsorption on tri-block copolymer Poloxamer 407 surface-modified microdialysis (MD) membranes were compared with protein adsorption on unmodified MD membranes. Ventricular cerebrospinal fluid (vCSF) kept at 37 °C was used as sample matrix. In total, 19 proteins were quantified in two biological replicates. The surface-modified membranes adsorbed 33% less proteins than control membranes and the most abundant proteins were subunits of hemoglobin and clusterin. The adsorption of clusterin on the modified membranes was on average 36% compared to control membranes. The most common protein in vCSF, Albumin, was not identified adsorbed to the surface at all. It was also experimentally verified that oSED, in conjunction with tandem mass spectrometry can be used to quantify femtomole amounts of proteins adsorbed on limited and delicate surfaces, such as MD membranes. The method has great potential and can be used to study much more complex protein adsorption systems than previously reported.

Analysis of membrane and hydrophilic proteins simultaneously derived from the mouse brain using cloud-point extraction.

In this study, a temperature-induced phase fractionation known as cloud-point extraction (CPE) with the non-ionic surfactant Triton X-114 was used to simultaneously extract, concentrate, and fractionate hydrophobic and hydrophilic proteins from mouse brain tissue. Two bottom-up proteomic techniques were used to comprehensively identify the extracted proteins. The first "shotgun"-based approach included tryptic digestion of the proteins followed by reversed-phase nanoliquid chromatography (RP-nanoLC) in combination with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). In the second approach, the extracted intact proteins were first separated by one-dimensional (1D) gel electrophoresis and then in-gel digested with trypsin and analyzed with nanoLC-MS/MS. In total, 1,825 proteins were unambiguously identified and the percentage of membrane proteins was 26% which is at the reported genome expression levels of 20-30%. The protein overlap between the two approaches was high. The majority (77%) of the identifications in the first approach was also found by the second method. The protein overlap between the CPE-extracted hydrophilic and hydrophobic fractions was rather small (16-23%) for both methods, which indicates a good phase separation. A quantitative evaluation of the CPE with iTRAQ labeling and nanoLC-ESI-MS/MS analysis gave iTRAQ ratios at the expected levels and an overall variation of the entire method at 17-31%. The results indicate very reproducible sample preparation and analysis methods that readily can be applied on large-scale sample sets.

Optimizing the extraction, separation and quantification of tricyclic antidepressant drugs in human plasma with CE-ESI-TOF-MS using cationic-coated capillaries.

In this study, the extraction and CE-ESI-TOF-MS analysis of tricyclic antidepressant (TCA) drugs imipramine, desipramine, clomipramine and norclomipramine in human plasma has been optimized. The CE capillaries were modified with ω-iodo-alkyl ammonium salt (M7C4I coating) to reduce analyte adsorption to the silica wall. The use of a strong cation exchange (SCX) solid-phase extraction (SPE) column specifically designed for the extraction of basic drug species from biofluids gave very clean extracts with high and reproducible recoveries. The extraction recoveries were ranging between 87 and 91% with % RSD values of 0.5-1.7% (n=3). The obtained strong cation exchange-SPE extracts of the TCA in human plasma only contained the analytes of interest. The optimized CE separation conditions were obtained by adding ACN and acetic acid to the sample while using an aqueous BGE. The CE-ESI-TOF-MS analysis was performed within 6 min for all TCA analytes under the optimized condition with peak efficiencies up to 1.4 x 105 plates/m and an average % RSD of the migration times of the analytes of 0.3% (n=5). The presented method can readily be used for the extraction and quantification of basic drug species in human biological fluids and in pharmaceutical formulations.

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.

Mining ventricular cerebrospinal fluid from patients with traumatic brain injury using hexapeptide ligand libraries to search for trauma biomarkers.

Traumatic brain injury (TBI) is an acute event resulting from external force to the brain and is a major cause of death and disability associated with high health care costs in the western world. Additional injuries, originating from the secondary molecular events after the initial intensive care, may be limited by the use of objective biomarkers to provide the best treatment and patient prediction outcome. In this study, hexapeptide ligand libraries (HLL) have been used for the enrichment of suggested protein biomarkers for TBI in cerebrospinal fluid (CSF). HLL have the potential to enrich low abundant proteins and simultaneously reduce the high abundant proteins, rendering a sample with significantly reduced dynamic range. The CSF proteome from two TBI inflicted patients have been extensively mapped using a large initial sample volume obtained by extraventricular drainage. Shotgun proteomics, in combination with isoelectric focusing (IEF) and nano-LC-MS/MS, identified 339 unique proteins (MudPIT scoring p < or = 0.05) with a protein overlap of 130 between the patients. As much as 45% of the proteins reported in the literature to be associated with degenerative/regenerative processes occurring after a trauma to the head were identified. Out of the most prominent potential protein biomarkers, such as neuron specific enolase, glial fibrillary acidic protein, myelin basic protein, creatine kinase B-type and S-100beta, all except myelin basic protein were detected in the study. This study shows the possibility of using HLL as a tool for screening of low abundant protein biomarkers in human CSF.

Assessment of the partitioning capacity of high abundant proteins in human cerebrospinal fluid using affinity and immunoaffinity subtraction spin columns.

The performance of three different affinity and immunoaffinity subtraction spin columns was investigated for the removal of the most abundant proteins in human cerebrospinal fluid (CSF). A pool of human CSF was processed with the spin columns and both the bound and flow through fractions were compared with each other and with intact CSF using 1D gel electrophoresis and nanoLC-MALDI-TOF/TOF-MS analysis. MASCOT MS/MS ionscores were compared before and after processing with the columns. The non-specific co-removal of proteins bound to the high abundant proteins, so called "sponge effect" was also examined for each spin column. The reproducibility of one of the spin columns, ProteomeLab IgY-12 proteome partitioning spin column, was further investigated by isobaric tags for relative and absolute quantification (iTRAQ) labeling and MS/MS analysis. Overall, 173 unique proteins were identified on a 95% MudPIT confidence scoring level. For all three spin columns, the number of proteins identified and their MASCOT scores were increased up to 10 times. The largest degree of non-specific protein removal was observed for a purely affinity based albumin removal column, where 28 other proteins also were present. The ProteomeLab IgY-12 proteome partitioning spin column showed very high reproducibility when combined with iTRAQ labeling and MS/MS analysis. The combined relative standard deviation (R.S.D.) for the high abundant protein removal, iTRAQ labeling and nanoLC-MALDI-TOF/TOF-MS analysis was less than 17.5%.

Methodological aspects on microdialysis protein sampling and quantification in biological fluids: an in vitro study on human ventricular CSF.

There is growing interest in sampling of protein biomarkers from the interstitial compartment of the brain and other organs using high molecular cutoff membrane microdialysis (MD) catheters. However, recent data suggest that protein sampling across such MD membranes is a highly complex process that needs to be further studied. Here, we report three major improvements for microdialysis sampling of proteins in complex biological matrixes. The improvements in this in vitro study using human ventricular cerebrospinal fluid as the sample matrix include increased fluid recovery control, decreased protein adsorption on the microdialysis membrane and materials, and novel quantitative mass spectrometry analysis. Dextrans in different concentrations and sizes were added to the perfusion fluid. It was found that dextrans with molecular mass 250 and 500 kDa provided a fluid recovery close to 100%. An improved fluid recovery precision could be obtained by self-assembly triblock polymer surface modification of the MD catheters. The modified catheters also delivered a significantly increased extraction efficiency for some of the investigated proteins. The final improvement was to analyze the dialysates with isobaric tagged (iTRAQ) proteomics, followed by tandem mass spectrometric analysis. By using this technique, 48 proteins could be quantified and analyzed with respect to their extraction efficiencies. The novel aspects of microdialysis protein sampling, detection, and quantification in biological fluids presented in this study should be considered as a first step toward better understanding and handling of the challenges associated with microdialysis sampling of proteins. The next step is to optimize the developed methodology in vivo.