Surface-enhanced Laser Desorption/ionization Mass Spectrometry Research Articles

A spherical covalent-organic framework for enhancing laser desorption/ionization mass spectrometry for small molecule detection.

A spherical vinyl-functionalized covalent-organic framework (COF-V) was prepared at room temperature by a facile method and applied as a novel substrate for surface-enhanced laser desorption/ionization mass spectrometry (SELDI-MS). Compared with conventional organic matrices, the spherical COF-V with high crystallinity and good monodispersity exhibited high sensitivity, no matrix background interference, wide-range applicability, high salt tolerance and reproducibility in the characterization of small molecules. Considering these advantages, the applicability of the spherical COF-V-based SELDI-MS method was successfully demonstrated by determining trace amounts of glucose in diabetic urine, which would be a promising candidate for clinical diagnosis of diabetes. In addition, the morphological effect and the desorption/ionization mechanism of the COF-V were investigated in detail and the results indicated that the spherical COF-V substrate could greatly enhance the LDI process compared with the bulk COF-V. This work not only extends the application of COFs in MS, but also offers a promising alternative for small molecule identification and clinical diagnosis of diabetes.

Nanoparticle-based surface assisted laser desorption ionization mass spectrometry: a review.

Great endeavors are undertaken to find effective nanoparticles to replace organic matrices for the analysis of small molecules using laser desorption ionization mass spectrometry (LDI-MS). Nanoparticles offer high sensitivity and better selectivity compared to conventional organic matrices. Surface assisted LDI-MS (SALDI-MS), and surface enhanced LDI-MS (SELDI-MS) provide clear background spectra without observable interferences peaks, and cause no fragmentation (soft ionization) of thermal and acidity labile molecules. This review article (with 460references) summarizes the recent applications of nanoparticles including metallic, metal oxides, silicon, quantum dots, metal-organic frameworks and covalent organic frameworks, for the analysis of small molecules. Nanoparticles serve not only as surface for LDI-MS, but they can be also used as probe or pseudo-stationary phase for separation, enrichment, and microextraction. Hopefully, the knowledge and learning points gained from this review will deepen the understanding of nanoparticles applications for LDI-MS. Graphical Abstract Schematic representation of laser desorption ionization mass spectrometry using various nanoparticles (such as metallic nanoparticles, carbon nanomaterials, silicon based nanomaterials, metal oxides, quantum dots, metal-organic frameworks, and covalent organic frameworks). Advanced technologies using nanoparticles are also reviewed.

Magnetic nanocomposite-based SELDI probe for extraction and detection of drugs, amino acids and fatty acids.

The use of carbon black-Fe3O4 magnetic nanocomposite (CB-Fe3O4) as a probe for surface-enhanced laser desorption ionization mass spectrometry (SELDI-MS) with a high extraction efficiency and sensitive detection is described. The magnetic nanocomposite was synthesized and fully characterized using X-ray photoelectron spectroscopy, X-ray diffraction, Raman spectroscopy, Fourier transform infrared spectroscopy, Ultraviolet-Visible spectroscopy, transmission electron microscopy and nitrogen sorption. The feasibility of the SELDI probe to extract and detect three classes of drugs (labetalol, metoprolol, doxepin, desipramine, triprolidine and methapyrilene) spiked in wine is demonstrated. All the drugs were successfully and reproducibly extracted and detected with high efficiency and with limits of detection (LOD) between 1 and 1000pgmL-1. The adsorption capacity of the nanocomposite for the drugs was evaluated by UV-Vis spectroscopy. The results showed that 27.8-36.1% of the drugs were adsorbed on the magnetic probe within 3min. The nanocomposite was also applied for efficient analysis of amino acids and fatty acids. Both types of analytes can be extracted within a few minutes and then successfully quantified by SELDI-MS. Graphical abstract A schematic presentation of carbon black-Fe3O4 magnetic probe for SELDI analysis of small molecules. The probe containing the analyte(s) is collected with the aid of a magnet and deposited on the target plate for mass spectrometry analysis.

Peptidyl-prolyl cis-trans isomerase A – A novel biomarker of multi-episodic (recurrent) ocular toxoplasmosis

Ocular toxoplasmosis (OT) is the most common etiology of posterior uveitis. The high incidence of macular scarring associated with OT is a leading cause of visual morbidity. Serum biomarkers of the disease would aid in its diagnosis. This study sought, for the first time, to elucidate serum biomarkers for OT by mass spectrometry. Blood samples were collected from four groups of nine patients each; toxoplasmosis IgG-with no history of uveitis, non-toxoplasmosis uveitis, first episode OT, and symptomatic recurrent OT. Serum was isolated and subjected to proteomics analysis using 2-dimensional gel electrophoresis (2D-GE) and surface-enhanced laser desorption ionization mass spectrometry (SELDI-MS). Selected proteins were further separated by SDS-PAGE and sequenced using tandem MS. Results were cross-validated with a T. gondii outbreak biomarker database that occurred in Brazil. Fifty markers of OT and 46 markers of recurrent disease were discovered by SELDI-MS of which 30 and 15, respectively, were cross-validated. 2D-GE analysis yielded 57 bands, selected based on the intensity of the bands, leading to the identification of 20 proteins. Eleven of those identified candidates were also found by SELDI-MS. Four candidates were chosen for immunoblotting. One serum protein, peptidyl-prolyl cis-trans isomerase A (PPIA), was confirmed as a biomarker of multi-episodic OT by immunoblotting in patients. PPIA can identify the patient with active recurrent OT from acute OT, other forms of uveitis and other parasitic infections. A validated PPIA assay may have a role in the diagnosis of the atypical OT patient before more invasive anterior chamber or vitreous tap is performed for PCR analysis or for Goldmann-Witner coefficient calculations. Base-line PPIA levels need to be studied to understand its possible use when deciding for prophylactic antibiotic use in the immunosuppressed sero-positive patient.

Magnetic nanoparticle modified chitosan for surface enhanced laser desorption/ionization mass spectrometry of surfactants

Chitosan (CTS) modified magnetic nanoparticles (CTS@Fe3O4MNPs) offer dual functions for the detection of surfactants using surface enhanced laser desorption/ionization mass spectrometry (SELDI-MS).

Identification & Characterization of Novel Hemostatic Biomarkers of Adverse Clinical Events in Patients with Continuous Flow Left Ventricular Assist Device Implants

Introduction: Heart failure affects over 5 million people in the US. Its rising prevalence coupled with a lack of donor hearts has led to an increase in the use of continuous flow left ventricular assist devices (CF-VAD). However, patients with CF-VAD implants are at risk for complications such as non-surgical bleeding and thrombosis, which significantly increase the risk of patient morbidity and mortality. Identification of novel biomarkers for these events could improve current risk assessment models, treatment, and thus, quality of life for VAD implanted patients.Materials and Methods: 71 blood samples were collected peri-operatively and at varying time points post-operatively from 16 patients implanted with a Thoratec HeartMate II CF-VAD. Plasma was prepared and stored at -70°C until analysis. Plasma samples collected from normal individuals were used as controls. Samples were analyzed for annexin V-positive microparticles and microparticles exposing tissue factor, as well as plasma levels of annexin V and C-reactive protein (CRP) by ELISA. Additionally, surface-enhanced laser desorption ionization - mass spectrometry (SELDI-MS) analysis was used to identify changes in biomarker expression. The occurrence of adverse clinical events was determined using an internal RedCap database. CF-VAD thrombosis was characterized as: cerebrovascular accident/transient ischemic attack diagnosed by a neurologist, rise in LDH or plasma free hemoglobin, hemolysis, evidence of pump dysfunction consistent with thrombus identified by pump parameters, echocardiographic or computed tomographic evidence of clot, or surgical pump exchange for thrombus. CF-VAD bleeding events were characterized as: anemia and bleeding determined by a cardiologist.Results: Annexin V plasma levels were increased approximately 3-fold compared to normal plasma (9.23 ±0.85 vs. 2.76 ±0.2 nM). CRP plasma levels were significantly elevated in CF-VAD patients compared to normals (14.5 ±2.7 vs. 2.2 ± 0.6 µg/ml; p=0.034). Although the level of annexin V-positive microparticles were the same in CF-VAD patients and normals, the amount of microparticles exposing tissue factor was significantly higher in CF-VAD patients (3.7 ± 1.1 vs. 0.5 ± 0.1 pg/ml; p=0.038). SELDI-MS analysis indicated three distinct peaks that were present in CF-VAD patients, but absent from normals (8.1, 11.7 and 15.2/16.2 kDa). During the follow-up period, patients experienced 12 CF-VAD-related thrombotic events, 8 hemorrhagic events and 2 septic events. The 8.1 kDa biomarker was present in 10/12 patients with a thrombotic event, 2/6 patients with a hemorrhagic event, 0/2 patients experiencing sepsis and 0/2 patients without an adverse event. Positive correlations with the SELDI-MS peaks and measured parameters were identified: the 8.1 kDa peak was associated with elevated plasma levels of annexin V (p=0.01) and the presence of annexin V-positive microparticles (p=0.005); the 11.7 kDa peak was associated with elevated plasma levels of CRP (p=0.01); the 15.2/16.2 kDa peaks were associated with the presence of microparticle exposing tissue factor (p=0.002), annexin V-positive microparticles (p<0.001) and elevated plasma levels of annexin V (p=0.03).Conclusions: Despite treatment with low-dose aspirin and warfarin, CF-VAD patients exhibit signs of hemostatic activation. SELDI-MS may be useful in the identification of novel biomarkers of CF-VAD-associated adverse events. This data justifies further evaluation of the utility of the SELDI-MS to readily identify biomarkers related to adverse clinical events in a larger CF-VAD population. DisclosuresNo relevant conflicts of interest to declare.

One pot synthesis of gold – carbon dots nanocomposite and its application for cytosensing of metals for cancer cells

Cytosensing of biological transition metals is paramount important for cancer research. One-pot synthesis of the nanocomposite of carbon dots (CDs) and gold nanoparticles (AuNPs) is reported and has been applied for metal cytosensing. The nanocomposite (GCDs) is synthesized using citric acid (CA) and L-cysteine as precursors in the presence of chloroauric acid. The synthesis procedure exhibits many advantages including simple one step, green approach (solvent free method) and requires cheap chemical precursors. The current procedure produces uniform carbon dots (<3nm) and assist the formation of AuNPs without using extra reducing agent. GCDs nanoparticles have UV absorbance matching with the wavelength of N2 laser (337nm). The synergistic effect of the large surface area of GCDs and the maximum absorbance at 337nm offers an effective and promising application for surface enhanced laser desorption/ionization mass spectrometry (SELDI-MS) of biological metals (Fe2+, Fe3+ and Cu2+ ions) for cancer cells. SELDI-MS using GCDs offers a sensitive method, shows selective detection, and provides simultaneous cytosensing of biological metals (Fe2+, Fe3+ and Cu2+) of cancer cells. The metals are detected after complexation with mefenamic acid (MFA) which provides multi-functions (chelating agent, internal calibrant and co-matrix) for LDI-MS.

The tumor suppressor ING1b is a novel corepressor for the androgen receptor and induces cellular senescence in prostate cancer cells.

The androgen receptor (AR) signaling is critical for prostate cancer (PCa) progression to the castration-resistant stage with poor clinical outcome. Altered function of AR-interacting factors may contribute to castration-resistant PCa (CRPCa). Inhibitor of growth 1 (ING1) is a tumor suppressor that regulates various cellular processes including cell proliferation. Interestingly, ING1 expression is upregulated in senescent primary human prostate cells; however, its role in AR signaling in PCa was unknown. Using a proteomic approach by surface-enhanced laser desorption ionization-mass spectrometry (SELDI-MS) combined with immunological techniques, we provide here evidence that ING1b interacts in vivo with the AR. The interaction was confirmed by co-immunoprecipitation, in vitro GST-pull-down, and quantitative intracellular colocalization analyses. Functionally, ING1b inhibits AR-responsive promoters and endogenous key AR target genes in the human PCa LNCaP cells. Conversely, ING1b knockout (KO) mouse embryonic fibroblasts (MEFs) exhibit enhanced AR activity, suggesting that the interaction with ING1b represses the AR-mediated transcription. Also, data suggest that ING1b expression is downregulated in CRPCa cells compared with androgen-dependent LNCaP cells. Interestingly, its ectopic expression induces cellular senescence and reduces cell migration in both androgen-dependent and CRPCa cells. Intriguingly, ING1b can also inhibit androgen-induced growth in LNCaP cells in a similar manner as AR antagonists. Moreover, ING1b upregulates different cell cycle inhibitors including p27(KIP1), which is a novel target for ING1b. Taken together, our findings reveal a novel corepressor function of ING1b on various AR functions, thereby inhibiting PCa cell growth.

A Novel Truncated Form of Serum Amyloid A in Kawasaki Disease.

BackgroundKawasaki disease (KD) is an acute vasculitis in children that can cause coronary artery abnormalities. Its diagnosis is challenging, and many cytokines, chemokines, acute phase reactants, and growth factors have failed evaluation as specific biomarkers to distinguish KD from other febrile illnesses. We performed protein profiling, comparing plasma from children with KD with febrile control (FC) subjects to determine if there were specific proteins or peptides that could distinguish the two clinical states.Materials and MethodsPlasma from three independent cohorts from the blood of 68 KD and 61 FC subjects was fractionated by anion exchange chromatography, followed by surface-enhanced laser desorption ionization (SELDI) mass spectrometry of the fractions. The mass spectra of KD and FC plasma samples were analyzed for peaks that were statistically significantly different.ResultsA mass spectrometry peak with a mass of 7,860 Da had high intensity in acute KD subjects compared to subacute KD (p = 0.0003) and FC (p = 7.9 x 10−10) subjects. We identified this peak as a novel truncated form of serum amyloid A with N-terminal at Lys-34 of the circulating form and validated its identity using a hybrid mass spectrum immunoassay technique. The truncated form of serum amyloid A was present in plasma of KD subjects when blood was collected in tubes containing protease inhibitors. This peak disappeared when the patients were examined after their symptoms resolved. Intensities of this peptide did not correlate with KD-associated laboratory values or with other mass spectrum peaks from the plasma of these KD subjects.ConclusionsUsing SELDI mass spectrometry, we have discovered a novel truncated form of serum amyloid A that is elevated in the plasma of KD when compared with FC subjects. Future studies will evaluate its relevance as a diagnostic biomarker and its potential role in the pathophysiology of KD.

A SELDI mass spectrometry study of experimental autoimmune encephalomyelitis: sample preparation, reproducibility, and differential protein expression patterns

BackgroundExperimental autoimmune encephalomyelitis (EAE) is an autoimmune, inflammatory disease of the central nervous system that is widely used as a model of multiple sclerosis (MS). Mitochondrial dysfunction appears to play a role in the development of neuropathology in MS and may also play a role in disease pathology in EAE. Here, surface enhanced laser desorption ionization mass spectrometry (SELDI-MS) has been employed to obtain protein expression profiles from mitochondrially enriched fractions derived from EAE and control mouse brain. To gain insight into experimental variation, the reproducibility of sub-cellular fractionation, anion exchange fractionation as well as spot-to-spot and chip-to-chip variation using pooled samples from brain tissue was examined.ResultsVariability of SELDI mass spectral peak intensities indicates a coefficient of variation (CV) of 15.6% and 17.6% between spots on a given chip and between different chips, respectively. Thinly slicing tissue prior to homogenization with a rotor homogenizer showed better reproducibility (CV = 17.0%) than homogenization of blocks of brain tissue with a Teflon® pestle (CV = 27.0%). Fractionation of proteins with anion exchange beads prior to SELDI-MS analysis gave overall CV values from 16.1% to 18.6%. SELDI mass spectra of mitochondrial fractions obtained from brain tissue from EAE mice and controls displayed 39 differentially expressed proteins (p≤ 0.05) out of a total of 241 protein peaks observed in anion exchange fractions. Hierarchical clustering analysis showed that protein fractions from EAE animals with severe disability clearly segregated from controls. Several components of electron transport chain complexes (cytochrome c oxidase subunit 6b1, subunit 6C, and subunit 4; NADH dehydrogenase flavoprotein 3, alpha subcomplex subunit 2, Fe-S protein 4, and Fe-S protein 6; and ATP synthase subunit e) were identified as possible differentially expressed proteins. Myelin Basic Protein isoform 8 (MBP8) (14.2 kDa) levels were lower in EAE samples with advanced disease relative to controls, while an MBP fragment (12. 4kDa), likely due to calpain digestion, was increased in EAE relative to controls. The appearance of MBP in mitochondrially enriched fractions is due to tissue freezing and storage, as MBP was not found associated with mitochondria obtained from fresh tissue.ConclusionsSELDI mass spectrometry can be employed to explore the proteome of a complex tissue (brain) and obtain protein profiles of differentially expressed proteins from protein fractions. Appropriate homogenization protocols and protein fractionation using anion exchange beads can be employed to reduce sample complexity without introducing significant additional variation into the SELDI mass spectra beyond that inherent in the SELDI- MS method itself. SELDI-MS coupled with principal component analysis and hierarchical cluster analysis provides protein patterns that can clearly distinguish the disease state from controls. However, identification of individual differentially expressed proteins requires a separate purification of the proteins of interest by polyacrylamide electrophoresis prior to trypsin digestion and peptide mass fingerprint analysis, and unambiguous identification of differentially expressed proteins can be difficult if protein bands consist of several proteins with similar molecular weights.

Profiling the Proteome of Exhaled Breath Condensate in Healthy Smokers and COPD Patients by LC-MS/MS

Three pools of exhaled breath condensate (EBC) from non-smokers plus healthy smokers (NS + HS, n = 45); chronic obstructive pulmonary disease (COPD) without emphysema (COPD, n = 15) and subjects with pulmonary emphysema associated with α1-antitrypsin deficiency (AATD, n = 23) were used for an exploratory proteomic study aimed at generating fingerprints of these groups that can be used in future pathophysiological and perhaps even clinical research. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was the platform applied for this hypothesis-free investigation. Analysis of pooled specimens resulted in the production of a “fingerprint” made of 44 proteins for NS/HS; 17 for COPD and 15 for the group of AATD subjects. Several inflammatory cytokines (IL-1α, IL-1β, IL-2; IL-12, α and β subunits, IL-15, interferon α and γ, tumor necrosis factor α); Type I and II cytokeratins; two SP-A isoforms; Calgranulin A and B and α1-antitrypsin were detected and validated through the use of surface enhanced laser-desorption ionization mass spectrometry (SELDI-MS) and/or by Western blot (WB) analysis. These results are the prelude of quantitative studies aimed at identifying which of these proteins hold promise as identifiers of differences that could distinguish healthy subjects from patients.

Naturally Occurring Human Urinary Peptides for Use in Diagnosis of Chronic Kidney Disease

Because of its availability, ease of collection, and correlation with physiology and pathology, urine is an attractive source for clinical proteomics/peptidomics. However, the lack of comparable data sets from large cohorts has greatly hindered the development of clinical proteomics. Here, we report the establishment of a reproducible, high resolution method for peptidome analysis of naturally occurring human urinary peptides and proteins, ranging from 800 to 17,000 Da, using samples from 3,600 individuals analyzed by capillary electrophoresis coupled to MS. All processed data were deposited in an Structured Query Language (SQL) database. This database currently contains 5,010 relevant unique urinary peptides that serve as a pool of potential classifiers for diagnosis and monitoring of various diseases. As an example, by using this source of information, we were able to define urinary peptide biomarkers for chronic kidney diseases, allowing diagnosis of these diseases with high accuracy. Application of the chronic kidney disease-specific biomarker set to an independent test cohort in the subsequent replication phase resulted in 85.5% sensitivity and 100% specificity. These results indicate the potential usefulness of capillary electrophoresis coupled to MS for clinical applications in the analysis of naturally occurring urinary peptides.

S100A7, a Novel Alzheimer's Disease Biomarker with Non-Amyloidogenic α-Secretase Activity Acts via Selective Promotion of ADAM-10

Alzheimer's disease (AD) is the most common cause of dementia among older people. At present, there is no cure for the disease and as of now there are no early diagnostic tests for AD. There is an urgency to develop a novel promising biomarker for early diagnosis of AD. Using surface-enhanced laser desorption ionization-mass spectrometry SELDI-(MS) proteomic technology, we identified and purified a novel 11.7-kDa metal- binding protein biomarker whose content is increased in the cerebrospinal fluid (CSF) and in the brain of AD dementia subjects as a function of clinical dementia. Following purification and protein-sequence analysis, we identified and classified this biomarker as S100A7, a protein known to be involved in immune responses. Using an adenoviral-S100A7 expression system, we continued to examine the potential role of S100A7 in AD amyloid neuropathology in in vitro model of AD. We found that the expression of exogenous S100A7 in primary cortico-hippocampal neuron cultures derived from Tg2576 transgenic embryos inhibits the generation of β-amyloid (Aβ)1–42 and Aβ1–40 peptides, coincidental with a selective promotion of “non- amyloidogenic” α-secretase activity via promotion of ADAM (a disintegrin and metalloproteinase)-10. Finally, a selective expression of human S100A7 in the brain of transgenic mice results in significant promotion of α-secretase activity. Our study for the first time suggests that S100A7 may be a novel biomarker of AD dementia and supports the hypothesis that promotion of S100A7 expression in the brain may selectively promote α-secretase activity in the brain of AD precluding the generation of amyloidogenic peptides. If in the future we find that S1000A7 protein content in CSF is sensitive to drug intervention experimentally and eventually in the clinical setting, S100A7 might be developed as novel surrogate index (biomarker) of therapeutic efficacy in the characterization of novel drug agents for the treatment of AD.

Oncoproteomics of hepatocellular carcinoma: from cancer markers' discovery to functional pathways

Hepatocellular carcinoma (HCC) is a heterogeneous cancer with no promising treatment and remains one of the most prevailing and lethal malignancies in the world. Researchers in many biological areas now routinely identify and characterize protein markers by a mass spectrometry-based proteomic approach, a method that has been commonly used to discover diagnostic biomarkers for cancer detection. The proteomic research platforms span from the classical two-dimensional polyacrylamide gel electrophoresis (2-DE) to the latest Protein Chip or array technology, which are often integrated with the MALDI (matrix-assisted laser-desorption ionization), SELDI (surface-enhanced laser desorption/ionization) or tandem mass spectrometry (MS/MS). New advances on quantitative proteomic analysis (e.g. SILAC, ICAT, and ITRAQ) and multidimensional protein identification technology (MudPIT) have greatly enhanced the capability of proteomic methods to study the expressions, modifications and functions of protein markers. The present article reviews the latest proteomic development and discovery of biomarkers in HCC that may provide insights into the underlying mechanisms of hepatocarcinogenesis and the readiness of biomarkers for clinical uses.

Dermokine: An Extensively Differentially Spliced Gene Expressed in Epithelial Cells

Studies performed to discover genes overexpressed in inflammatory diseases identified dermokine as being upregulated in such disease conditions. Dermokine is a gene that was first observed as expressed in the differentiated layers of skin. Its two major isoforms, alpha and beta, are transcribed from different promoters of the same locus, with the alpha isoform representing the C terminus of the beta isoform. Recently, additional transcript variants have been identified. Extensive in silico analysis and reverse transcriptase (RT)-PCR cloning has confirmed the existence of these variants in human cells and tissues, identified a new human isoform as well as the gamma isoform in mouse. Recombinant expression and analysis of the C-terminal truncated isoform indicate that the molecule is O-linked glycosylated and forms multimers in solution. In situ hybridization and immunohistochemistry has shown that the gene is differentially expressed in various cells and tissues, other than the skin. These results show that the dermokine gene is expressed in epithelial tissues other than the skin and this expression is transcriptionally and posttranscriptionally complex.

Comparison of Computational Algorithms for the Classification of Liver Cancer using SELDI Mass Spectrometry: A Case Study

Introduction As an alternative to DNA microarrays, mass spectrometry based analysis of proteomic patterns has shown great potential in cancer diagnosis. The ultimate application of this technique in clinical settings relies on the advancement of the technology itself and the maturity of the computational tools used to analyze the data. A number of computational algorithms constructed on different principles are available for the classification of disease status based on proteomic patterns. Nevertheless, few studies have addressed the difference in the performance of these approaches. In this report, we describe a comparative case study on the classification accuracy of hepatocellular carcinoma based on the serum proteomic pattern generated from a Surface Enhanced Laser Desorption/Ionization (SELDI) mass spectrometer. Methods Nine supervised classification algorithms are implemented in R software and compared for the classification accuracy. Results We found that the support vector machine with radial function is preferable as a tool for classification of hepatocellular carcinoma using features in SELDI mass spectra. Among the rest of the methods, random forest and prediction analysis of microarrays have better performance. A permutation-based technique reveals that the support vector machine with a radial function seems intrinsically superior in learning from the training data since it has a lower prediction error than others when there is essentially no differential signal. On the other hand, the performance of the random forest and prediction analysis of microarrays rely on their capability of capturing the signals with substantial differentiation between groups. Conclusions Our finding is similar to a previous study, where classification methods based on the Matrix Assisted Laser Desorption/Ionization (MALDI) mass spectrometry are compared for the prediction accuracy of ovarian cancer. The support vector machine, random forest and prediction analysis of microarrays provide better prediction accuracy for hepatocellular carcinoma using SELDI proteomic data than six other approaches.

Identification of Novel Peptide Safety Markers for Exocrine Pancreatic Toxicity Induced by Cyanohydroxybutene

Historically, serum amylase and lipase levels have been used to indicate pancreas injury; however, these enzyme levels are often not predictive of pathology. In an effort to discover novel biomarkers of pancreatic acinar cell injury, we analyzed serum and pancreas tissue from cyanohydroxybutene (CHB)-treated male IGS rats using proteomics methods. CHB produces an "edematous pancreatitis," characterized by depletion of zymogen granules, acinar cell apoptosis, and mild to moderate inflammation. Secondary necrosis occurs at higher doses. Rats were treated with 150 mg/kg of CHB and samples were collected at 4, 8, and 24 h. Analyses of serum tryptic digests by surface-enhanced laser desorption-ionization mass spectrometry revealed two novel peptide biomarkers (RA1609 and RT2864) that were predictive of pancreatic damage. Levels of RA1609 decreased, while levels of RT2864 increased by 8 h following CHB treatment. The changes in RA1609 and RT2864 were detected in media from CHB-treated primary rat acini, demonstrating that these peptides are either of pancreatic cell origin or are produced by proteases released from acinar cells. Sequencing revealed that RA1609 is a fragment of rat albumin (accession number P02770, residues 348-360) and RT2864 is a portion of either rat trypsin III (accession number P08426, residues 39-65) or bovine trypsin (accession number P00760, residues 35-61). These two peptides, and possibly other fragments of serum proteins that are digested by pancreatic proteases, may be useful as safety markers for exocrine pancreatic toxicity during drug development or as biomarkers for the diagnosis and/or grading of severity of pancreatic disease.

Expression optimization and purification process development of an engineered soluble recombinant mouse linker of activation of T cells using surface enhanced laser desorption/ionization-mass spectrometry

Protein purification development is the bottleneck of recombinant protein production therefore there is a need to shorten process development and monitoring. Surface enhanced laser desorption/ionization-mass spectrometry (SELDI-MS) was evaluated to optimize the expression and to develop the purification of a recombinant mouse protein: a transmembrane adaptor involved in T cell receptor signaling named “linker for activation of T cells” (LAT). The protein was expressed as a soluble form (S-LAT) in three strains of Escherichia coli: BL21 ( DE3), Rosetta ( DE3), and BL21 ( DE3) pLys S. The expression of S-LAT was monitored on immobilized metal affinity chromatography (IMAC) ProteinChip ® arrays. The highest level of expression was found in Rosetta ( DE3) with a C-terminal construct after induction at 37 °C. The purification scheme was elucidated using SELDI-MS: S-LAT was efficiently captured on an IMAC ProteinChip array saturated with nickel ions (Ni 2+) and then fractionated on a Q ProteinChip array. These conditions were directly transferred to IMAC-Ni 2+ HyperCel and Q Ceramic HyperD ® F chromatography sorbents. After these two purification steps, S-LAT was estimated to be more than 80% pure, confirming a very good match between array and sorbent. Finally, a peptide mapping was performed on a hydrophobic array after in gel trypsin digest, verifying that the purified protein was the mouse LAT. This is the first report of a protocol for the production and purification of S-LAT. The selection of the best expression and purification strategy along with the identification were enabled in 5 days with less than 5 mL of soluble fraction of crude culture samples.

Reducing protein concentration range of biological samples using solid-phase ligand libraries☆

The discovery of specific polypeptides of diagnostic relevance from a biological liquid is complicated by the overall vast number and the large concentration range of all polypeptides/proteins in the sample. Depletion or fractionation methodologies have been used for selectively removing abundant proteins; however, they failed to significantly enrich trace proteins. Here we expand upon a new method that allows the reduction of the protein concentration range within a complex mixture, like neat serum, through the simultaneous dilution of high abundance proteins and the concentration of low abundance ones in a single, simple step. This methodology utilizes solid-phase ligand libraries of large diversity. With a controlled sample-to-ligand ratio it is possible to modulate the relative concentration of proteins such that a large number of peptides or proteins that are normally not detectable by classical analytical methods become, easily detectable. Application of this method for reducing the dynamic range of unfractionated serum is specifically described along with treatment of other biological extracts. Analytical surface enhanced laser desorption/ionization mass spectrometry (SELDI-MS) technology and mono- and two-dimensional electrophoresis (1-DE and 2-DE) demonstrate the increase in the number of proteins detected. Examples linking this approach with additional fractionation methods demonstrate a further increase in the number of detectable species using either the so-called "top down" or "bottom up" approaches for proteomics analysis. By enabling the detection of a greater proportion of polypeptides/proteins within a sample, this method may contribute significantly towards the discovery of new biomarkers of diagnostic relevance.

Immunoproteomic characterization of Brucella abortus 1119-3 preparations used for the serodiagnosis of Brucella infections

The diagnosis of brucellosis is mainly based on the detection of anti-LPS antibodies. Due to substantial similarity of the O-polysaccharide of Brucella LPS to that of various other Gram-negative bacteria, serological tests of samples containing high amounts of LPS lack specificity. Hence, the development of assays based on more specific protein antigens is an essential subject in brucellosis research. The aim of this study was proteomic characterization of various antigen preparations of the diagnostic reference strain Brucella abortus 1119-3 and the identification of immunogenic proteins suitable for serological assays. Seventeen out of 383 protein spots of B. abortus 1119-3 were identified to be immunogenic by 2-D immunoblotting. These immunogenic spots were assigned to 6 proteins by MALDI-MS and nLC-ESI-MS/MS: Cu–Zn SOD, BCSP31, L7/L12, GroEL, GroES, and DnaK. All immunogenic proteins were present in three different antigen preparations investigated, i.e. native antigen, standard agglutination and commercially available agglutination antigen. 2-D immunoblotting of bacteria cross-reacting with Brucellae in agglutination tests proved that cross-reactivity of proteins is negligible. Surface enhanced laser desorption/ionization mass spectrometry (SELDI-MS) spectra also differentiated B. abortus clearly from cross-reacting bacteria. The combination of SELDI-MS analysis with the specificity of antibody binding will improve the identification of Brucella specific immunogenic proteins.