From SELDI-TOF MS to protein identification by on-chip elution

SELDI-TOF MS has been demonstrated as a powerful tool for biomarker discovery. However, a major disadvantage of SELDI-TOF MS is the lack of direct identification of the discriminatory peaks discovered. We describe a novel experimental identification strategy where peptides/proteins captured to a weak cation exchange ProteinArray surface (CM10) are eluted, and thereafter identified by utilizing a sensitive LC-MS/MS (i.e. LTQ Orbitrap). A mixture of four known proteins was used to test the novel experimental approach described, and all four proteins were successfully identified. Additionally, a biomarker candidate previously discovered in plasma of Atlantic cod (Gadus morhua) by SELDI-TOF MS was identified. Thus, this study indicated that a combination of on-chip elution and a highly sensitive LC-MS/MS system can be an alternative approach to identify biomarker candidates discovered by use of SELDI-TOF MS.     

Proteomic characterization of inter-alpha inhibitor proteins from human plasma

Inter-alpha inhibitor proteins (IaIp) are a family of structurally related serine protease inhibitors found in relatively high concentrations in human plasma. Recent studies have implicated a role for IaIp in sepsis, and have demonstrated their potential as biomarkers in sepsis and cancer. For characterization of isolated IaI proteins and contaminating proteins during the last steps of the purification process, SELDI-TOF MS and HPLC-ESI-MS/MS were used. After separation by SDS-PAGE or 2-DE, polypeptide bands of 80, 125 and 250 kDa were excised from gels and digested by trypsin. The tryptic peptides were analyzed by both MS methods. The main contamination during the purification process, a band of 80 kDa, contains mainly IaIp heavy chain (HC) H3. HC H1 and H2 were also found in this band. In addition, some vitamin K-dependent clotting factors and inhibitors and other plasma proteins were identified. The 125-kDa band, representing the pre-alpha inhibitor, was found to contain both bikunin and HC H3. The presence of other HC H1, H2 and the recently described HC H4 was also detected by SELDI-TOF MS. The presence of HC H1, H2, and H3 in the 125-kDa band was confirmed by ESI-MS/MS, but not the presence of the H4. Three polypeptides, H1 and H2 together with bikunin, were identified in the 250-kDa band, representing the ITI, by both MS techniques. Once again, the presence of H4 was detected in this band only by SELDI-TOF MS, but the number of corresponding peptides was still not sufficient for final identification of this polypeptide. The importance of the application of proteomic methods for the proper evaluation of therapeutic drugs based on human plasma is discussed.     

A novel method for analyzing phosphoproteins using SELDI-TOF MS in combination with a series of recombinant proteins

A novel SELDI-TOF MS-based method for analyzing phosphoproteins was developed using a series of recombinant wild-type and mutant ribosomal P2 proteins. We demonstrated that the phosphorylation status of the overexpressed proteins in cells was easily and rapidly confirmed using this method. The ribosomal P2 protein contained two phosphorylation sites, which were sequentially phosphorylated in vivo. We also quantitatively detected the phosphoprotein by using SELDI-TOF MS.     

The nucleotide-independent Fe(III)-binding site is located on beta subunit of the mitochondrial F(1)-ATPase

The need for methods to identify disease biomarkers is underscored by the survival-rate of patients diagnosed at early stages of cancer progression. Surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) is a novel approach to biomarker discovery that combines two powerful techniques: chromatography and mass spectrometry. One of the key features of SELDI-TOF MS is its ability to provide a rapid protein expression profile from a variety of biological and clinical samples. It has been used for biomarker identification as well as the study of protein-protein, and protein-DNA interaction. The versatility of SELDI-TOF MS has allowed its use in projects ranging from the identification of potential diagnostic markers for prostate, bladder, breast, and ovarian cancers and Alzheimer's disease, to the study of biomolecular interactions and the characterization of posttranslational modifications. In this minireview we discuss the application of SELDI-TOF MS to protein biomarker discovery and profiling.     

Detection and identification of plasma proteins that bind GlialCAM using ProteinChip arrays, SELDI-TOF MS, and nano-LC MS/MS

In order to fully understand biological processes it is essential to identify interactions in protein complexes. There are several techniques available to study this type of interactions, such as yeast two-hybrid screens, affinity chromatography, and coimmunoprecipitation. We propose a novel strategy to identify protein-protein interactions, comprised of first detecting the interactions using ProteinChips and SELDI-TOF MS, followed by the isolation of the interacting proteins through affinity beads and RP-HPLC and finally identifying the proteins using nano-LC MS/MS. The advantages of this new strategy are that the primary high-throughput screening of samples can be performed with small amounts of sample, no specific antibody is needed and the proteins represented on the SELDI-TOF MS spectra can be identified with high confidence. Furthermore, the method is faster and less labor-intensive than other current approaches. Using this novel method, we isolated and identified the interactions of two mouse plasma proteins, mannose binding lectin C and properdin, with GlialCAM, a type 1 transmembrane glycoprotein that belongs to the Ig superfamily.     

Differential analysis of protein expression of Bifidobacterium grown on different carbohydrates

We observed recently that colonic fermentation of lactose might be a major factor in the pathophysiology of lactose intolerance. Proteomic techniques could be helpful in interpreting the metabolic pathways of lactose fermentation. The objective of this study was to explore proteomic methodologies for studying bacterial lactose metabolism that can be used to detect and identify proteins associated with the onset of intolerance symptoms. Differential expression of cytoplasmic proteins of Bifidobacterium animalis, Bifidobacterium breve and Bifidobacterium longum grown on different carbohydrates (lactose, glucose, galactose) was analyzed with surface-enhanced laser desorption ionization-time of flight (SELDI-TOF) MS and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After fractionation by SDS-PAGE, differentially-expressed proteins were identified with LC-MS/MS. The three strains grown on the same carbohydrate or the same strain grown on glucose or lactose showed differences in SELDI-TOF MS protein profiles. Differences in protein expression were observed in B. breve grown on glucose, galactose or lactose as analyzed with SDS-PAGE. With LC-MS/MS, proteins from Bifidobacterium were identified, which included enzymes for metabolism of lactose, glucose and galactose. In conclusion, the applied techniques can discern differences in protein expression of bacteria metabolizing different carbohydrates. These techniques are promising in studying metabolism of lactose and other substrates in a complex bacterial ecosystem such as the colonic microbiota.     

Identification of plasma protein biomarkers associated with idiopathic pulmonary arterial hypertension

We have employed SELDI-TOF MS to screen for differentially expressed proteins in plasma samples from 27 patients with idiopathic pulmonary arterial hypertension (IPAH) and 26 healthy controls. One ion (m/z approximately 8600) that was found to be elevated in IPAH was validated by SELDI-TOF MS analysis of a second and separate set of plasma samples comprising 30 IPAH patients and 19 controls. The m/z 8600 was purified from plasma by sequential ion exchange and reverse-phase chromatographies and SDS-PAGE. It was identified, following trypsin digestion, by MS peptide analysis as the complement component, complement 4a (C4a) des Arg. Plasma levels of C4a des Arg measured by ELISA confirmed that the levels were significantly higher (p < 0.0001) in IPAH patients (2.12 +/- 0.27 microg/mL) compared with normal controls (0.53 +/- 0.05 microg/mL). A cut-off level of 0.6 microg/mL correctly classified 92% of IPAH patients and 80% of controls. Further studies will be needed to determine its performance as a diagnostic biomarker, whether used alone or in combination with other biomarkers. Nevertheless, this study demonstrates that putative biomarkers characteristic of IPAH can be identified using a conjoint SELDI-TOF MS - proteomics approach.     

SELDI-TOF MS Proteomics in Breast Cancer

Background

Proteomic profiling is a rapidly developing technology that may enable early disease screening and diagnosis. Surface-enhanced laser desorption ionization–time of flight mass spectrometry (SELDI-TOF MS) has demonstrated promising results in screening and early detection of many diseases. In particular, it has emerged as a high-throughput tool for detection and differentiation of several cancer types. This review aims to appraise published data on the impact of SELDI-TOF MS in breast cancer.

Methods

A systematic literature search between 1965 and 2009 was conducted using the PubMed, EMBASE, and Cochrane Library databases. Studies covering different aspects of breast cancer proteomic profiling using SELDI-TOF MS technology were critically reviewed by researchers and specialists in the field.

Results

Fourteen key studies involving breast cancer biomarker discovery using SELDI-TOF MS proteomic profiling were identified. The studies differed in their inclusion and exclusion criteria, biologic samples, preparation protocols, arrays used, and analytical settings. Taken together, the numerous studies suggest that SELDI-TOF MS methodology may be used as a fast and robust approach to study the breast cancer proteome and enable the analysis of the correlations between proteomic expression patterns and breast cancer.

Conclusion

SELDI-TOF MS is a promising high-throughput technology with potential applications in breast cancer screening, detection, and prognostication. Further studies are needed to resolve current limitations and facilitate clinical utility.     

Proteomic analysis of fast and slow muscles from normal and kyphoscoliotic mice using protein arrays, 2-DE and MS

A proteomic strategy based upon the integrated use of SELDI-TOF/MS, 2-DE and MALDI-TOF/MS has been used to identify a panel of fast muscle protein markers: MLC1F, MLC3F, fast troponin C (STNC) and slow muscle markers: MLC1SB and MLC2v. MLC3F, MLC1F and STNC were virtually absent in the physiologically pure slow soleus muscle of kyphoscoliotic mutant mice compared to control BDmice, whereas MLC2v increased threefold. A SELDI-TOF/MS peak at 18,012 Da in spectra from strong anionic exchange protein array fractions of fast vastus muscle was confirmed as STNC by its specific depletion from crude extracts of vastus muscle using an anti-TNC mAb. SELDI-TOF/MS also identified MLC2F phosphorylation in crude muscle extracts after treatment with alkaline phosphatase. High probability protein identifications were achieved by SELDI-TOF/MS PMF based upon the resolution of large peptides formed by partial cleavage and high peptide coverage. When the pI from 2-D gels and molecular weight estimations from SELDI-TOF/MS were entered into the TagIdent algorithm, high probability protein identity predictions were obtained that were confirmed later by PMF. We confirm that SELDI-TOF/MS can be integrated with other proteomics techniques for the efficient analysis of protein expression changes and PTMs associated with physiological changes in skeletal muscle.     

Efficient monitoring of enzymatic conjugation reaction by surface-enhanced laser desorption/ionization time of flight mass spectrometry for process optimization

Efficient analysis of bioconjugation reactions is one the most challenging task for optimizing and eventually achieving the reproducible production of large amount of conjugates. In particular, the complexity of some reaction mixtures precludes the use of most of the existing methods, because of the presence of large amounts of contaminants. As an alternative method, we used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) for monitoring an in vitro enzymatic transglycosylation of N-acetylgalactosamine (GalNAc) residues to a recombinant mucin protein MUC6. For this reaction, catalyzed by the uridine 5'-diphospho-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts), we used either a recombinant ppGalNAc-T1 or a mixture of ppGalNAc-Ts contained in MCF7 tumor cell extracts. In the present study, we show that SELDI-TOF MS offers unique advantages over the traditional methodologies. It is a rapid, accurate, sensitive, reproducible, and very convenient analytical method for monitoring the course of a bioconjugation, even in heterogeneous samples such as cell extracts. SELDI-TOF MS proved very useful for optimizing the reaction parameters of the transglycosylation and for achieving the large scale preparation of Tn antigen-glycosylated mucins for antitumor immunotherapy applications.     

Identification of treatment efficacy-related host factors in chronic hepatitis C by ProteinChip serum analysis

Recent development of proteomic array technology, including protein profiling coupling ProteinChip array with surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF/MS), provides a potentially powerful tool for discovery of new biomarkers by comparison of its profiles according to patient phenotypes. We used this approach to identify the host factors associated with treatment response in patients with chronic hepatitis C (CHC) receiving a 48-wk course of pegylated interferon (PEG-IFN) alpha 2b plus ribavirin (RBV). Protein profiles of pretreatment serum samples from 32 patients with genotype 1b and high viral load were conducted by SELDI-TOF/MS by using the three different ProteinChip arrays (CM10, Q10, IMAC30). Proteins showed significantly different peak intensities between sustained virological responders (SVRs), and non-SVRs were identified by chromatography, SDS-PAGE, TOF/MS and tandem mass spectrometry (MS/MS) assay. Eleven peak intensities were significantly different between SVRs and non-SVRs. The three SVR-increased peaks could be identified as two apolipoprotein (Apo) fragments and albumin and, among the eight non-SVR-increased proteins, four peaks identified as two iron-related and two fibrogenesis-related protein fragments, respectively. Multivariate analysis showed that the serum ferritin and three peak intensity values (Apo A1, hemopexin and transferrin) were independent variables associated with SVRs, and the area under the receiver operating characteristic (ROC) curves for SVR prediction by using the Apo A1/hemopexin and hemopexin/transferrin were 0.964 and 0.936. In conclusion, pretreatment serum protein profiling by SELDI-TOF/MS is variable for identification of response-related host factors, which are useful for treatment efficacy prediction in CHC receiving PEG-IFN plus RBV. Our data also may help us understand the mechanism for treatment resistance and development of more effective antiviral therapy targeted toward the modulation of lipogenesis or iron homeostasis in CHC patients.     

Evaluation of the SELDI-TOF MS technique for protein profiling of pancreatic islets exposed to glucose and oleate

The aim of the study was to evaluate the SELDI-TOF MS technique for pancreatic islet research. Mouse islets were cultured at low or high glucose levels in the absence or presence of oleate and characterized by measuring insulin secretion and oxygen tension. Subsequently, the islets were protein profiled. Up to 200 different peaks could be detected in a single experiment with the majority of peaks corresponding to proteins with masses below 30 kDa. By combining different protein arrays, the number of detected peaks could be increased further. The optimal binding of islet proteins was achieved using the anionic exchange array and phosphate buffer (pH 6) when the binding of insulin was low, which allowed other less abundant proteins to be captured. When islets from different culture conditions were profiled and analyzed, in total 25 proteins were found to be oleate/glucose-regulated. An oleate-regulated protein was chosen for identification work, which was conducted by passive elution from SDS-PAGE gels and subsequent in-gel trypsin digestion and MALDI-TOF MS. The protein was identified as peptidyl-prolyl isomerase B (PPI-B). In conclusion, the study demonstrates that SELDI-technique can be used not only to obtain islet protein patterns but is also helpful in the subsequent identification of differentially expressed proteins.     

Identification of complement C3a as a candidate biomarker in human chronic hepatitis C and HCV-related hepatocellular carcinoma using a proteomics approach

Although the significant risk factors for hepatocellular carcinoma (HCC) are well known from epidemiological studies, diagnosis of this disease at an early stage is difficult, and HCC remains one of the leading causes of cancer death worldwide. Thus, to identify any useful HCC-related biomarkers is still a need. We performed SELDI-TOF MS to identify differentially expressed proteins in HCC serum using weak cation exchange protein chips. Protein characterization was performed by 2-DE separation and nano flow LC-MS/MS. A total of 55 sera were collected from HCC patients and compared with those from 48 patients with chronic hepatitis and 9 healthy individuals. A candidate marker of about 8900 Da was detected as differentially expressed in patients with chronic hepatitis C and hepatitis C virus (HCV)-related HCC. We identified this differentially expressed protein as complement C3a. The expression of C3a in HCC sera was further validated by PS20 chip immunoassay and Western blotting. Complement C3a was found to be elevated in patients with chronic hepatitis C and HCV-related HCC. The combination of SELDI-TOF MS and 2-DE provides a solution to identify disease-associated serum biomarkers.     

蛋白质芯片SELDI-TOFMS技术的研究进展及其在临床中的应用

蛋白质芯片为新一代的蛋白质组研究技术,由美国Ciphergen生物系统公司引进,表面增强激光解吸电离-飞行时间质谱(SELDI-TOFMS)提供一个高通量和高灵敏度的检测平台。投放至今虽短短10来年,但卓越的成果已广为医学科学界重视,尤其在恶性肿瘤的早期诊断、监控和预后研究上。蛋白质是细胞内执行生物功能的最终分子,蛋白质组学研究让人类更深入了解疾病和生命的本源,不断发现的特异性肿瘤标志物更为攻克癌症带来新希望。这里除对表面增强激光解吸电离_飞行时间质谱作较详尽的介绍外,更重点阐述其近年来蛋白质芯片近期的研究进展和在临床中的应用,并就其优劣和发展前景作出评估。     

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.     

Identification of alphaB-crystallin, a biomarker of renal cell carcinoma by SELDI-TOF MS

Spectrometric-based surface-enhanced laser desorption/ionization ProteinChip (SELDI-TOF) facilitates rapid and easy analysis of protein mixtures and is often exploited to define potential diagnostic markers from sera. However, SELDI- TOF is a relatively insensitive technique and unable to detect circulating proteins at low levels even if they are differentially expressed in cancer patients. Therefore, we applied this technology to study tissues from renal cell carcinomas (RCC) in comparison to healthy controls. We found that different biomarkers are identified from tissues than those previously identified in serum, and that serum markers are often not produced by the tumors themselves at detectable levels, reflecting the nonspecific nature of many circulating biomarkers. We detected and characterized áB-crystallin as an overexpressed protein in RCC tissues and showed differential expression by immunohistochemistry. We conclude that SELDI-TOF is more useful for the identification of biomarkers that are synthesized by diseased tissues than for the identification of serum biomarkers and identifies a separate set of markers. We suggest that SELDI-TOF should be used to screen human cancer tissues to identify potential tissue-specific proteins and simpler and more sensitive techniques can then be applied to determine their validity as biomarkers in biological fluids.     

Limitations in quantitation of the biomarker CCL18 in Gaucher disease blood samples by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry

SELDI-TOF MS assisted the discovery of the chemokine CCL18/PARC as plasma biomarker for pathological storage cells in Gaucher disease patients. Prognostic elevation of CCL18 in blood of Gaucher patients has been confirmed by ELISA. Given its low molecular mass, positive charge, and relatively high abundance, CCL18 seems a particular attractive protein for SELDI-TOF based quantitation. Therefore, we determined CCL18 levels in plasma using SELDI-TOF MS and ELISA, in parallel. CCL18 levels in some blood samples were significantly underestimated when determined by SELDI-TOF MS. Spiking of recombinant CCL18 indicated that its detection by SELDI-TOF MS is strongly determined by the nature of the sample, even markedly varying between samples obtained from one donor at different time points. Independent of the total CCL18 concentration in blood only 1-10% of the chemokine bound to the ProteinChip Array. Even when comparable amounts of CCL18 from distinct samples were bound to the ProteinChip Array, diverse peak intensities could be observed. Thus, limited binding capacity and sample-dependent suppression of CCL18 ionization contribute significantly to the final peak intensity. In conclusion, SELDI-TOF MS offers no reliable procedure to quantitatively monitor CCL18 levels in blood and thus cannot be applied in evaluation of disease status of Gaucher patients.     

The application of SELDI-TOF mass spectrometry to mammalian cell culture

Surface Enhanced Laser Desorption/Ionisation Time-of-Fight Mass Spectrometry (SELDI-TOF MS) is a technique by which protein profiles can be rapidly produced from a wide variety of biological samples. By employing chromatographic surfaces combined with the specificity and reproducibility of mass spectrometry it has allowed for profiles from complex biological samples to be analysed. Profiling and biomarker identification have been employed widely throughout the biological sciences. To date, however, the benefits of SELDI-TOF MS have not been realised in the area of mammalian cell culture. The advantages in identifying markers for cell stresses, apoptosis and other culture parameters mean that these tools could help greatly to enhance monitoring and control of bioreaction process and improve the production of therapeutics. Better characterisation of culture systems through proteome analysis will allow for improved productivity and better yields.     

Identification of serum proteomic biomarkers for early porcine reproductive and respiratory syndrome (PRRS) infection

ABSTRACT: BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant swine diseases worldwide. Despite its relevance, serum biomarkers associated with early-onset viral infection, when clinical signs are not detectable and the disease is characterized by a weak anti-viral response and persistent infection, have not yet been identified. Surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF MS) is a reproducible, accurate, and simple method for the identification of biomarker proteins related to disease in serum. This work describes the SELDI-TOF MS analyses of sera of 60 PRRSV-positive and 60 PRRSV-negative, as measured by PCR, asymptomatic Large White piglets at weaning. Sera with comparable and low content of hemoglobin (< 4.52 ug/mL) were fractionated in 6 different fractions by anion-exchange chromatography and protein profiles in the mass range 1-200 kDa were obtained with the CM10, IMAC30, and H50 surfaces. RESULTS: A total of 200 significant peaks (p < 0.05) were identified in the initial discovery phase of the study and 47 of them were confirmed in the validation phase. The majority of peaks (42) were up-regulated in PRRSV-positive piglets, while 5 were down-regulated. A panel of 14 discriminatory peaks identified in fraction 1 (pH=9), on the surface CM10, and acquired at low focus mass provided a serum protein profile diagnostic pattern that enabled to discriminate between PRRSV-positive and -negative piglets with a sensitivity and specificity of 77% and 73%, respectively. CONCLUSIONS: SELDI-TOF MS profiling of sera from PRRSV-positive and PRRSV-negative asymptomatic piglets provided a proteomic signature with large scale diagnostic potential for early identification of PRRSV infection in weaning piglets. Furthermore, SELDI-TOF protein markers represent a refined phenotype of PRRSV infection that might be useful for whole genome association studies.     

Improving feature detection and analysis of surface-enhanced laser desorption/ionization-time of flight mass spectra

Discovering valid biological information from surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS) depends on clear experimental design, meticulous sample handling, and sophisticated data processing. Most published literature deals with the biological aspects of these experiments, or with computer-learning algorithms to locate sets of classifying biomarkers. The process of locating and measuring proteins across spectra has received less attention. This process should be tunable between sensitivity and false-discovery, and should guarantee that features are biologically meaningful in that they represent chemical species that can be identified and investigated. Existing feature detection in SELDI-TOF MS is not optimal for acquiring biologically relevant data. Most methods have so many user-defined settings that reproducibility and comparability among studies suffer considerably. To address these issues, we have developed an approach, called simultaneous spectrum analysis (SSA), which (i) locates proteins across spectra, (ii) measures their abundance, (iii) subtracts baseline, (iv) excludes irreproducible measurements, and (v) computes normalization factors for comparing spectra. SSA uses only two key parameters for feature detection and one parameter each for quality thresholds on spectra and peaks. The effectiveness of SSA is demonstrated by identifying proteins differentially expressed in SELDI-TOF spectra from plasma of wild-type and knockout mice for plasma glutathione peroxidase. Comparing analyses by SSA and CiphergenExpress Data Manager 2.1 finds similar results for large signal peaks, but SSA improves the number and quality of differences betweens groups among lower signal peaks. SSA is also less likely to introduce systematic bias when normalizing spectra.