ProteinChip Surfaces Research Articles

Volunteer study and serum protein profiling to understand inflammatory response induced by Satsuma mandarin

It has been observed that consumption of a certain amount of Satsuma, lychee, and longan often caused a symptom characterized by dry or sore throat, gum swelling and even mouth ulcer, which significantly impaired the life quality of a large population. We define the adverse reaction to Satsuma as Satsuma-induced syndrome (SIS). Volunteers were assigned to oral Satsuma challenge in an open manner. The results showed that SIS was characterized with symptoms affecting the throat, oral cavity, face, gastrointestinal system and eye either individually or in combination. A comparative proteomic study was performed to investigate the differences of serum proteins in the Post-SC (after Satsuma challenge) and Pre-SC (before Satsuma challenge) serum samples of 15 volunteers with severe SIS. Ten proteins were identified to be differentially expressed (P<0.05). Of these, levels of complement component C9 precursor were elevated significantly in the Post-SC serum samples and were further verified by enzyme-linked immunosorbent assay, indicating that the complement system may be activated and plays a significant role in inflammatory response. Meanwhile, serum samples were subjected to immobilized metal affinity capture (IMAC3) protein chip surfaces and tested by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry. The data were analyzed by Ciphergen ProteinChip Software. A diagnostic model was constructed to discriminate the SIS from normal samples, using principal component analysis. A total of 50 detected biomarkers were found to be different with statistical significance (P<0.05). The multivariate logistic analysis demonstrates a complete distinction between the two groups. Our findings suggest that these assays may provide potential biomarkers for the diagnosis of SIS.

SELDI-TOF-MS Serum Profiling Reveals Predictors of Cardiac MRI Changes in Marathon Runners

Purpose. To utilize proteomics to discover proteins associated with significant cardiac magnetic resonance imaging (MRI) changes in marathon runners. Methods. Serum from 25 runners was analyzed by surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Proteomic profiles were compared in serum samples obtained prior to the race, at the finish line and within 7 hours after race to identify dynamic proteins correlated with cardiac MRI changes. Results. 693 protein/peptide clusters were identified using two ProteinChip surface chemistries and, of these, 116 were significantly different between the three time points. We identified 7 different patterns of protein expression change within the runners and 5 prerace protein peaks, 16 finish-line protein levels, and 15 postrace proteins which were correlated with significant postrace cardiac MRI changes. Conclusions. This study has identified baseline levels of proteins which may be predictive of risk of significant cardiac damage following a marathon race. Preliminary identification of the significant proteins suggested the involvement of cytokines and other proteins involved in stress and inflammatory response.

Regeneration of IMAC-Cu Proteinchip by Borate Eluent

Influence of borate eluent pH value and competition eluant on regeneration of the used IMAC-Cu proteinchip is investigated by SEM and SELDI-TOF-MS proteinchip detection system. The results show that proteins are easier to be removed from the used proteinchip surface with the decreasing of borate eluent pH value, but its destruction to the chemical structure of the proteinchip also grows in intensity. The elution effect of protein on IMAC-Cu proteinchip can be significantly enhanced when the competition eluant Gly or EDTA is added to borate eluate. However, comparing with Gly, EDTA as competition eluant has generation effect. The absorption protein quantity and type of the regenerated IMAC-Cu proteinchip by the pH 8 borate eluent added 0.02mol/L EDTA for 72h is very similar to the fresh chip after absorbing same serum of cancer patients.

Adiponectin Receptor-1 C-Terminal Fragment (CTF) in Plasma: Putative Biomarker for Diabetes

Abstract Introduction Polypeptide fragments from cell surface receptors when found in plasma may be indicators of receptor regulation in disease conditions. It is known that subjects with diabetes have significantly lower plasma concentrations of adiponectin, a hormone released by adipose tissue, compared with nondiabetic controls. This hormone interacts with cell surface receptors in muscle (AdipoR1) and liver (AdipoR2). Methods We analyzed the relative distribution of specific fragments of AdipoR1 in healthy and diabetic individuals using an immunoaffinity mass spectrometry approach. We used antibodies raised against AdipoR1 immobilized on pre-activated protein chip surfaces to determine the molecular weights of bound polypeptide fragments using immunomass spectrometry (immuno-MS). Results Initially, immuno-MS analyses using a polyclonal antibody revealed two peaks (m/z 3,902 and 7,812) in plasma from normal, healthy individuals (n = 5) that were not present in the plasma of diabetics (n = 5). To confirm the detection of these fragments, a monoclonal antibody was developed against the last 25 amino acids of the AdipoR1 C-terminal fragment (CTF). Using the immuno-MS method, the monoclonal antibody detected the AdipoR1 CTF (m/z 3475) in all healthy controls (n = 10), but did not detect these fragments in the diabetic patients (n = 10). Discussion These preliminary observations suggest that the plasma levels of this receptor fragment may serve as an indicator of diabetic condition.

Combined proteomic and RNA microarray analysis after TRIzol extraction of eutopic endometrium for endometriosis biomarker discovery

OBJECTIVE: Endometriosis (endo) is an important gynaecological disease defined by the presence of ectopic endometrium (EM) outside the uterus and associated with pelvic pain and infertility. In this study we evaluated for the first time the feasibility of EM TRIzol extraction to obtain both a protein and mRNA fraction of sufficient quality from the same EM sample in order to perform combined proteomic analysis and mRNA microarray.DESIGN: Proteomic analysis of TRIzol-extracted eutopic endometrium.MATERIALS AND METHODS: Endometrial tissues were obtained during the late luteal phase (day 23-26) from 16 patients including controls with a normal pelvis (n=5) and patients with endometriosis (n=11) including minimal-mild (n=5) and moderate-severe (n=6) disease according to ASRM classification. We used Surface Enhanced Laser Desorption/Ionisation Time-of-Flight Mass Spectrometry (SELDI-TOF-MS): all samples were measured in duplicates on CM10 and IMAC30 protein chips in low molecular weight (CHCA) and high molecular weight (SPA). All mass peaks were used as input for a Least Squares Support Vector Machine (LS-SVM). Next, Leave one out cross validation (LOO-CV) was used to estimate independent test set performance.RESULTS: Profiling of endometrial samples by using different ProteinChip surfaces showed differential expression of proteins and peptides in the range between 7 - 17 kDa depending on chip and matrix type. The LOO-CV performance of the LS-SVM models indicated an Area under the ROC curve (AUC) between 85%-100% (depending on chip and matrix type) for the diagnosis of endometriosis. These results will be discussed in relationship to the mRNA microarray results obtained on the same EM samples (Sherwin et al, 2008).CONCLUSIONS: Protein profiling using ProteinChip array after TRIzol preparation is feasible and reproducible and allows combined proteomic analysis and mRNA microarray on the same EM sample. OBJECTIVE: Endometriosis (endo) is an important gynaecological disease defined by the presence of ectopic endometrium (EM) outside the uterus and associated with pelvic pain and infertility. In this study we evaluated for the first time the feasibility of EM TRIzol extraction to obtain both a protein and mRNA fraction of sufficient quality from the same EM sample in order to perform combined proteomic analysis and mRNA microarray. DESIGN: Proteomic analysis of TRIzol-extracted eutopic endometrium. MATERIALS AND METHODS: Endometrial tissues were obtained during the late luteal phase (day 23-26) from 16 patients including controls with a normal pelvis (n=5) and patients with endometriosis (n=11) including minimal-mild (n=5) and moderate-severe (n=6) disease according to ASRM classification. We used Surface Enhanced Laser Desorption/Ionisation Time-of-Flight Mass Spectrometry (SELDI-TOF-MS): all samples were measured in duplicates on CM10 and IMAC30 protein chips in low molecular weight (CHCA) and high molecular weight (SPA). All mass peaks were used as input for a Least Squares Support Vector Machine (LS-SVM). Next, Leave one out cross validation (LOO-CV) was used to estimate independent test set performance. RESULTS: Profiling of endometrial samples by using different ProteinChip surfaces showed differential expression of proteins and peptides in the range between 7 - 17 kDa depending on chip and matrix type. The LOO-CV performance of the LS-SVM models indicated an Area under the ROC curve (AUC) between 85%-100% (depending on chip and matrix type) for the diagnosis of endometriosis. These results will be discussed in relationship to the mRNA microarray results obtained on the same EM samples (Sherwin et al, 2008). CONCLUSIONS: Protein profiling using ProteinChip array after TRIzol preparation is feasible and reproducible and allows combined proteomic analysis and mRNA microarray on the same EM sample.

Optimization of SELDI-TOF protein profiling for analysis of cervical mucous

Cervical mucous, produced in the region where cervical neoplasia occurs, is thought to be a good choice for discovery of biomarkers to improve cervical cancer screening. In this study, SELDI-TOF MS analysis was used to evaluate parameters for protein profiling of mucous. Proteins were extracted from mucous collected with Weck-Cel sponges. Several parameters like extraction reagent, loading protein concentration, matrix type, bind/wash conditions and sample fractionation, on different protein chip surfaces were evaluated. SELDI peak number and consistency in the resulting spectra were used to evaluate each condition. Analysis of spectra generated by different protein chips revealed an average of 30 peaks in the 2.5-30 kDa mass range using sinnapinic acid in the unfractionated sample. Sample concentration and buffer conditions evaluated did not lead to large alterations in the profiles. Quality control spectra were reproducible with intra- and inter-assay intensity CV for CM10, H50 and Q10 arrays being less than 20% and 30% respectively. IMAC30-Cu chips had higher intra- and inter-assay CV's at 25% and 35%. Current data showed that optimizing pre-analytical parameters can help in standardization and reproducibility of protein profiles produced by cervical mucous, and thus can be used for protein biomarker discovery with the SELDI platform.

A comparative analysis of polyurethane hydrogel for immobilization of IgG on chips

Hydrogels are considered an optimum material for protein chip surfaces, since they provide a quasi-liquid environment which allows protein activity to be maintained and shows good spot morphology as well as excellent immobilization capacity. In the following, we present a polyurethane (PU) chip that electrostatically binds IgG. The PU surface is optimized with regard to layer thickness (∼200 nm), hydrogel (2%) and immobilized antibody concentration (0.5 mg mL −1; 0.3 ng spot −1), pH and ionic strength of the print buffer as well as to blocking solution. Evaluation is done in a direct IgG immunoassay using the Nexterion slide H as a reference. It is shown that higher IgG loading is achieved on the PU chip than on slide H, no matter whether 1× PBS (pH 7.2), Sörensen (pH 5.8) or Nexterion buffer was used as a spotting solution. Moreover, the crossreactivity with goat IgG, human IgG and monoclonal anti-CRP spotted in Nexterion buffer was as low as ≤0.74% (slide H: ≤3.34%).

-Plasma analysis of rheumatoid arthritis by SELDI-

To identify protein biomarkers linking to disease activity and treatment responses of patients with rheumatoid arthritis (RA), proteomic study using mass spectrometric analysis of plasma proteins was performed. Proteomic profiling technologies can simultaneously resolve and analyze multiple proteins in plasma. Evaluation of multiple proteins of the plasma will be essential to discover protein biomarkers. In this study, we used protein chip surface enhanced laser desorption/ionization time of flight mass spectrometry approach (SELDI-TOF MS). Through differential profiling of plasma proteins, we selected two prospective candidate biomarkers. One mass spectrometric peak distinguished patients with RA from healthy controls was transthyretin (TTR) and the other distinguished inactive patients with RA from patients with active RA was Serum Amyloid A (SAA). This study demonstrates that proteomic profiling using mass spectrometry of plasma greatly facilitates global discovery and verify clinically relevant sets of disease biomarker directly links to disease activity and treatment responses.

Region-specific alterations of global protein expression in the remodelled rat myocardium

We applied the novel ProteinChip technology (SELDI-MS) to investigate and identify differentially regulated proteins upon myocardial remodelling in different heart regions. Tissue samples were isolated from the atria, the interventricular septum, and the right and left ventricles three months after surgically-induced myocardial infarction (MI) in rats. Corresponding protein extracts from control versus MI hearts were analysed on two different ProteinChip surfaces. In each of the functionally distinct cardiac regions, we obtained specific protein profile alterations upon myocardial remodelling. Most alterations were observed in the non-infarcted right ventricle, where down-regulation occurred more frequently than up-regulation of protein expression. Three of the differentially regulated proteins were identified: the metabolic enzyme triosephosphate isomerase (TIM), the cell signalling protein Raf-1 kinase inhibitory protein (RKIP), also known as phosphatidylethanolamine binding protein (PEBP), and the small heat shock protein alphaB-crystallin. These proteins showed a pronounced tissue-dependent regulation. TIM was down-regulated only in the atrium and in the left ventricle, RKIP/PEBP was down-regulated only in the right ventricle and in the interventricular septum, and alphaB-crystallin was up-regulated only in the right and in the left ventricle. A simple correlation of peak intensity changes using two of the identified peaks demonstrated the diagnostic potential of SELDI-MS. We conclude that this novel proteomic method is a powerful high-throughput tool for the fast detection of region-specific cardiac protein profiles in small biopsy samples, and that SELDI-MS may become a useful complementary technique for the diagnosis and prognosis of cardiac diseases.

Immunological evaluation of urinary trypsin inhibitors in blood and urine: Role of N- &amp; O-linked glycoproteins

Urinary trypsin inhibitors (uTi) suppress serine proteases during inflammation. After liberation from proinhibitors (P-alpha-I and I-alpha-I) by the white blood cell (WBC) response, uTi readily pass through the kidneys into urine. A key uTi, bikunin, is attached to O-linked and N-linked glycoconjugates. Recently, uTi inhibitors, called uristatins, were found to lack the O-linked glycoconjugates. Monoclonal antibodies were produced using purified uristatin and screened for binding differences to uristatin, bikunin, P-alpha-I, and I-alpha-I. Antibody-binding patterns were characterized using immunoaffinity binding onto protein-chip surfaces and analysis by Surface Enhanced Laser Desorption/Ionization mass spectrometry (SELDI), using specimens from patients and from purified uTi standards. Antibodies were developed and used in an enzyme-linked immunosorbent assay (ELISA) method for uTi measurement in urine and plasma specimens. ELISA was performed on specimens from normal, presumed healthy, controls and from patients who had been screened for inflammation using a high sensitivity C-reactive protein (CRP) test and a complete blood count (CBC). Polyclonal antibody against uTi showed cross-reactivity with the Tamm-Horsfall protein (THP) and with proinhibitors. Screening of anti-uTi monoclonal antibodies (Mab) revealed antibodies that did not cross-react with either of the above, thus providing a tool to measure both uristatin and bikunin in urine with Mab 3G5 and in plasma with Mab 5D11. The monoclonal antibody 5D11 cross-reacts with specific N-linked glycoconjugates of uristatin present in plasma. In ca 96% of healthy adults, uTi were present at <12 mg/l in urine and <4 mg/l in plasma. We also found that patients with an inflammation and a CRP of >2.0 mg/l had higher urinary concentrations of uTi than the control population in every subject. Free uristatin and bikunin pass readily into urine and are primarily bound to heavy chains that constitute the proinhibitor form in plasma.

Prediction of renal allograft rejection by urinary protein analysis using ProteinChip Arrays (surface-enhanced laser desorption/ionization time-of-flight mass spectrometry)

Objectives To develop a noninvasive method for the detection of renal transplant rejection using ProteinChip Arrays (surface-enhanced laser desorption/ionization time-of-flight mass spectrometry). Methods A total of 23 urine samples were collected from 13 patients showing biopsy-proven renal allograft rejection and from 10 patients without histologic signs of rejection. All 23 patients had clinical symptoms and signs of acute allograft rejection and underwent renal biopsy. Samples were centrifuged, and supernatants were directly spotted onto the ProteinChip arrays with different chromatographic surfaces. The obtained spectra in a range from 2 to 200 kDa were subjected to bioinformatic analysis using the method of Fuzzy c-means, followed by the establishment of rule bases and evaluation using the relevance index according to Kiendl. Results Several protein peaks were identified allowing differentiation between rejection and no rejection. Using two different ProteinChip surfaces, we found two biomarkers at 25.71 kDa and 28.13 kDa that gave a diagnostic sensitivity of 90% and 93% and a specificity of 80% (SAX2) and 85% (CM10), respectively. Conclusions Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry appears to be a promising new diagnostic tool for distinguishing renal transplant patients with no rejection from those with acute rejection.

25 SELDI-TOF-MS ProteinChip profiling of serum and nasal cells from Cystic Fibrosis patients

Surface enhanced laser desorption/ionisation-time of flight (SELDI-TOF) mass spectrometry (MS) is an array based proteomic technology. It combines a variety of chromatographic surfaces that bind proteins from biological mixtures according to physicochemical properties with MS analysis. The relative expression of proteins at specific molecular weights can then be compared among different samples. The aim of this work is to establish protein profiles in Cystic Fibrosis (CF) serum and airway cell lysate for biomarker identification. These proteins may serve as diagnostic/prognostic markers or even as new targets for CF therapy. Serum and nasal epithelial cells (collected by brushing) were obtained from CF patients (n 13/15) and controls (n 11/9). Protein extracts were applied to a range ProteinChip surfaces (anionic exchange, cationic exchange and metal affinity) and analysed by SELDI-TOF-MS. The mass spectral data demonstrate a larger group of proteins with differential expression in serum compared to nasal cells lysate. 45 peaks (p < 0.05) differentiated CF serum from control. 9 peaks (p < 0.05) differentiated CF nasal cell lysate from control. Further work is required to validate these results in a larger cohort of CF patients and to identify candidate biomarkers with MS protein characterisation. Work supported by FCT/FEDER research grants (POCTI/SAU-MMO/56163/2004). PA is a recipient of FCT doctoral fellowship. CB and DP collaborated equally to supervision of the experiments. 2. CFTR Cell biology~Physiology

Proteomic Identification of Dysregulated Apolipoprotein Expression in Pulmonary Hypertension Secondary to Sickle Cell Disease.

Pulmonary hypertension (PHT) is emerging as a major complication and independent risk factor for sudden death among adults with sickle cell disease (SCD). The identification of novel plasma markers of PHT might allow for earlier diagnosis and more convenient screening than offered by the current use of Doppler echocardiography to identify patients with high tricuspid regurgitant jet velocity (TRV). We used surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF MS) to search for plasma biomarkers of PHT from clinical specimens. A cohort of 27 homozygous SCD patients with PHT (mean TRV=3.1±0.4 m/sec) was compared with a group of 28 homozygous SCD patients without PHT (mean TRV=1.4±0.5 m/sec), selected for similar age, gender and renal function. Plasma samples were fractionated on anion exchange columns by step pH elution (pH 3–9, organic) and applied to solid-phase protein chip surfaces (Ciphergen Biosystems ProteinChips). Univariate and multivariate analysis of SELDI-TOF data was accomplished by Random Forest classification/regression (RF), stepwise logistical regression, and Spearman correlations with clinically measured parameters. The identity of significant protein species was confirmed through MALDI-TOF and or LC-MS/MS. Results and conclusions: SELDI-TOF MS analysis resulted in 28 sets of 162 spectra each, resolving 955 peaks per patient. RF and stepwise logistical regression modeling of these peaks consistently showed lower abundance of a 28.1 kDa protein in PHT patients (p<0.0001), identified by MALDI-TOF MS and LC-MS/MS as the oxidant scavenging protein apolipoprotein A-I (apo A-I). The 28.1 kDa intensities also correlated strongly with clinical assays of apo A-I (r=0.58, p<0.0001) and HDL levels (r=0.50, p=0.0001). Comparison of PHT prevalence between patients whose apo A-I levels fell in the lowest quartile indicated a risk ratio (RR) of 2.0 compared to the upper quartile (p=0.06). Patients in the upper quartile of apo B levels had a higher prevalence of PHT than the lowest quartile (RR 2.3, p=0.05), with even higher risk indicated by a high ratio of apo B/apo A-I (RR 3.3, p=0.006), all known to be risk indicators in cardiovascular disease without SCD. In addition, a 13.4 kDa peak, identified as the inflammatory marker serum amyloid A-4 (SAA-4) by LC-MS/MS, was also important by RF and was abundant in the PHT positive group (p=0.0006). An 8.9 kDa peak, significant in combination with 28.1 kDa by stepwise logistical regression (ROC area=0.88; p=0.007) was identified by LC-MS/MS as apolipoprotein A-II. These results implicate the apolipoprotein pathway in the development of PHT vasculopathy in sickle cell disease. Their known function in regulating oxidative species supports the proposed role of oxidant stress in the sickle cell vasculopathy. Our findings are consistent with published data indicating that an apo A-I mimetic dramatically improves blood flow in the sickle cell mouse. Further studies of these apolipoproteins are warranted to validate their potential mechanistic involvement in sickle vasculopathy and as predictive markers of PHT.

Proteomic Pattern Profiling of the Eutopic Endometrial and Ovarian Endometriotic Tissues in Same Patients Using SELDI-TOF Mass Spectroscopy

Endometriosis is thought to be a major factor for female infertility at reproductive ages. Despite of improvements in diagnosis and therapy, the treatment rate is poor so far. For a better understanding of the molecular mechanisms behind the process of endometriosis, we have analyzed the changes of protein expression pattern between the eutopic endometrial and matched ovarian endometriotic tissues in same patients by using a protein chip surface enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectroscopy.

Macro/Nano-Structured Silicon as Solid Support for Antibody Arrays: Surface Design, Reproducibility, and Assay Characteristics Enabling Discovery of Kallikrein Gene Products for Prostate Disease Diagnostics

To facilitate high-throughput biomarker discovery and high-density protein-chip array analyses of complex biological samples, a novel macro-and nanoporous silicon surface for protein microarrays was developed. The surface offers three-dimensional surface enlarging properties and spot confinement, enabling both high sensitivity bioassays and design of high density arrays. Reproducible manufacturing of the protein chip surface was accomplished as demonstrated by the low imprecision when standard IgG bioassays were performed at 100 pM antigen level on a series of protein chips scanned at widely different locations within a silicon wafer, as well as between different wafers from two different manufacturers. The relative standard deviation (RSD) of fluorescence spot intensity within an array on a chip was less than 20%. Mean spot intensity RSD was 19% for all 25 microarray chips in the study. Within-manufacturer-lot RSDs in chips from either manufacturer were <15% of mean spot intensity. The detection limit and dynamic range of the novel protein chip surface were examined to evaluate whether they match criteria required in a search for novel biomarkers such as for prostate cancer. Monoclonal IgG against prostate-specific antigen (PSA) was arrayed on the porous silicon chips. These were subsequently incubated in serum samples containing widely different levels of fluorescence-labeled PSA. Detection of PSA in serum at concentrations from 0.7 ng/mL (26 pM) up to 104-fold higher levels verified assay characteristics required in the search for prostate biomarkers (e.g., kallikrein gene products) at clinically relevant levels.

Bioinformatic Analysis of the Urine Proteome of Acute Allograft Rejection

The urinary proteome in health and disease attracts increasing attention because of the potential diagnostic and pathophysiologic biomarker information carried by specific excreted proteins or their constellations. This cross-sectional study aimed to analyze the urinary proteome in patients with biopsy-proven acute rejection (n = 23) compared with transplant recipients with stable graft function (n = 22) and healthy volunteers (n = 20) and to correlate this with clinical, morphologic, and laboratory data. Urine samples were preadsorbed on four different protein chip surfaces, and the protein composition was analyzed using a surface-enhanced laser desorption/ionization time-of-flight mass spectrometer platform. The data were analyzed using two independent approaches to sample classification. Patients who experienced acute rejection could be distinguished from stable patients with a sensitivity of 90.5 to 91.3% and a specificity of 77.2 to 83.3%, depending on the classifier used. Protein masses that were important in constructing the classification algorithms included those of mass 2003.0, 2802.6, 4756.3, 5872.4, 6990.6, 19,018.8, and 25,665.7 Da. Normal urine was distinguished from transplant urine using a protein marker of mass 78,531.2 Da with both a sensitivity and a specificity of 100%. In conclusion, (1) urine proteome in transplant recipients with stable graft function was significantly different from healthy control subjects, and (2) acute rejections were characterized by a constellation of excreted proteins. Analysis of the urinary proteome may expedite the noninvasive prediction of acute graft rejection, thus importantly assisting in establishing the diagnosis.

Identification of RIZ1 Targets Involvedin Erythroid Differentiation of K562 Human Erythroleukemia Cells Using Surface-Enhanced Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (SELDI).

RIZ1 (PRDM2) is a member of the nuclear protein methyltransferase superfamily involved in chromatin remodeling. RIZ1 functions as a tumor suppressor gene in a number of human cancers and is down regulated in some human acute myeloid leukemias. We previously found RIZ-1 to be silenced in K562 erythroleukemia cells by promoter hypermethylation. Furthermore, expression of RIZ1 in K562 promotes erythroid differentiation and also potentiates TGF-β1 mediated differentiation. To investigate similarities between genes altered by RIZ1 expression and the TGF-β1 pathway, we used SELDI to compare the protein profiles of K562 against K562 + RIZ1 and K562 + TGF-β1. Protein extracts for SELDI profiling were separated into six fractions according to their isoelectric points. Proteins from each fraction were then bound to two different protein chip surfaces (H50-hydrophoboic and CM10-cation exhange) and their mass/charge determined using SELDI. We analyzed four replicates from each sample and classified proteins as differentially expressed if their P-values were below 0.05. In total, we observed 104 differentially expressed proteins (60 upregulated and 44 down regulated) between K562 and K562 + RIZ1 and 176 proteins (96 upregulated and 80 down regulated) between K562 and K562 + TGF-β1. We used 2D-PAGE to identify differentially expressed proteins identified by SELDI analysis and located 48 proteins that were over expressed in K562 + RIZ1 and K562 + TGF-β1 relative to K562. To establish whether these proteins were the same proteins observed using SELDI, we determined if the proteins had the same pI and molecular weight and if the gel-eluted proteins bound to the same protein chip surface with the same mass/charge. 15 of 48 proteins passed the above criteria and we determined their identities using Trypsin-based peptide mapping strategies with molecular weight and pI restrictions. We identified two candidate proteins (14-3-3ε and S100/A13) that are similarly over expressed in K562 + RIZ1 and K562 + TGF-β1. These proteins have been shown to be associated with TGF-β1 signaling. Schistosomal 14-3-3ε interacts with SmRK1, a divergent type I transforming growth factor β1 receptor (TR-I) present on the surface of adult parasites and also binds to and activates human TR-I. S100/A13 belongs to a family of low molecular weight proteins characterized by the presence of two calcium-binding EF-hand motifs that includes S100C/A11, a member recently shown to play a key role in a PKCα mediated pathway essential for the growth inhibition of normal human keratinocytes by TGF-β1. In summary, we demonstrate the potential for using SELDI to identify novel proteins involved in regulating and connecting cellular growth and differentiation pathways.

A Pilot Application of SELDI Mass Spectrometry To Study Proteomic Patterns in Plasma from Patients with T-Cell Large Granular Lymphocyte Leukemia.

T-cell large granular lymphocyte (T-LGL) leukemia is a rare chronic lymphoproliferative disorder of cytotoxic T cells of unknown etiology. Chronic antigenic stimulation of T-lymphocytes in autoimmune diseases or viral infection has been postulated to play a role in the pathogenesis of this condition. Surface-enhanced laser desorption/ionization (SELDI) mass spectrometry of plasma and serum specimens was applied as a useful screening method to determine pathophysiologic protein/peptide markers, which may be useful in the diagnosis of occult malignancies. In contrast to traditional diagnostic principles, the SELDI method enables screening of protein patterns without knowing the identity of targets. Patterns of up- and down-modulation of peptides and proteins (resulting from the presence of abnormal products of diseased cells, pathogens or immunogenetic factors) may signal various clinical features and diagnostic clues. We used SELDI mass spectrometry to study proteomic patterns in the plasma of patients with T-LGL leukemia. After initial screening and optimization we selected a hydrophobic H4 ProteinChip surface for further systematic screening. Deviation of mass values and peak intensities were <0.5% and <20%, respectively.Reproducibility was monitored by random duplication of samples with an average of 70 peaks per spectrum (mass/charge ratio from 1500–50,000 Da). Our study included plasma samples from 28 LGL patients, 9 patients with idiopathic neutropenia (without the presence of LGL clone) and 36 healthy blood donors. The diagnosis of T-LGL leukemia included the presence of monoclonal TCR rearrangement and flow cytometric detection of abnormal CD8+CD3+CD57+ cell population. Spectra were analyzed using Biomarker Patterns Software, which allows for internal cross validation for the prediction of the accuracy in a blinded test set. To separate LGL patients from patients with chronic neutropenia, intensity cut-off for 2050 Da peak was found in 22/28 LGL patients, resulting in 100% specificity and 67% sensitivity. Based on the intensities of 3 mass peaks (2274, 5870, 2660 Da), the sensitivity and specificity of LGL distinction from healthy controls was 96% and 61%, respectively. By direct digestion with carboxypeptidase Y on the ProteinChip surface, the C-terminal sequence of the 2050 Da mass peak has been identified. From the sequencing analysis and the search in the ExPASy protein database, we concluded that the 2050 Da mass peak corresponded to the fragment of complement C3 precursor. Additional 2 kDa mass peaks may represent various other C3 fragments. Interestingly, the intensity of the mass peak corresponding to the C3 fragments decreased with the application of immunosuppressive therapy when plasma samples from 3 LGL patients were serially analyzed. The pathophysiologic significance of the identified peak remains unclear, but increased expression of the C3 receptor on LGL cells has been reported. Our data in LGL leukemia indicate that SELDI mass spectrometry can be used to identify immune biomarkers for malignant lymphoproliferations.

Serum diagnosis of pancreatic adenocarcinoma using surface-enhanced laser desorption and ionization mass spectrometry.

Each year in the United States, approximately 30,000 people die from pancreatic cancer. Fewer than 5% of patients survive >5 years after diagnosis, because most patients present with advanced disease. Early diagnosis may improve the prognosis of patients with pancreatic cancer. In an attempt to improve on current approaches to the serological diagnosis of pancreatic cancer, we analyzed serum samples from patients with and without pancreatic cancer using surface-enhanced laser desorption and ionization (SELDI) protein chip mass spectrometry. Using a case-control study design, serum samples from 60 patients with resectable pancreatic adenocarcinoma were compared with samples from 60 age- and sex-matched patients with nonmalignant pancreatic diseases, as well as 60 age- and sex-matched healthy controls. To increase the number of proteins potentially identifiable, serum was fractionated using anion exchange and profiled on two ProteinChip surfaces (metal affinity capture and weak cation exchange). We determined a minimum set of protein peaks able to discriminate between patient groups and used the unified maximum separability algorithm to compare the performance of the individual marker panels alone or in conjunction with CA19-9. Among the peaks identified by SELDI profiling that had the ability to distinguish between patient groups, the 2 most discriminating protein peaks could differentiate patients with pancreatic cancer from healthy controls with a sensitivity of 78% and specificity of 97%. These 2 markers performed significantly better than the current standard serum marker, CA19-9 (P < 0.05). The diagnostic accuracy of the 2 markers was improved by using them in combination with CA 19-9. Similarly, a combination of 3 SELDI markers and CA19-9 was superior to CA19-9 alone in distinguishing individuals with pancreatic cancer from the combined pancreatic disease controls and healthy subject groups (P = 0.078). SELDI markers were also better than CA19-9 in distinguishing patients with pancreatic cancer from those with pancreatitis. SELDI profiling of serum can be used to accurately differentiate patients with pancreatic cancer from those with other pancreatic diseases and from healthy controls.

Methods for on-chip protein analysis

The unambiguous identification of peptides/proteins is crucial for the definition of the proteome. Using ProteinChip Array technology also known as surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS), we developed experimental protocols and probed test conditions required for the protein identification on ProteinChip surfaces. We were able to directly digest peptides/proteins on-chip surfaces by specific proteases, such as trypsin, and to obtain the peptide mass fingerprint of the sample under investigation by its direct analysis on a simple laser desorption/ionization mass spectrometer. Furthermore, tandem mass spectrometry was performed on several of the resulting tryptic peptides by using collision quadrupole time of flight (Qq-TOF) MS/MS via the ProteinChip interface, thus allowing the unambiguous identification of the protein(s) within the sample. In addition, we were able to identify the C-terminal sequence of peptides by their digestion with carboxypeptidase Y directly on ProteinChip surfaces coupled with SELDI-TOF MS analysis of the resulting peptide mass ladders employing the instrument’s protein ladder sequence software. Moreover, the removal of up to nine amino acid residues from the C-terminal end of a peptide extends the functional range of Qq-TOF MS/MS sequence determination to over 3000 m/ z. The utility of these procedures for the proteome exploration are discussed.