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PubMed2004

Proteomics for nasal secretion analysis.

Since the completion of the human genome, the interest of the scientific community has evolved toward understanding the human proteome. The genomic and proteomic data will facilitate our understanding of the functions of proteins in diseases and the discovery of novel drug target proteins and biomarkers of diseases. Highly sensitive analytic techniques are necessary to study the complexity of biologic samples. The key to any proteomics experiment is to reduce the complexity of the sample before mass spectrometry (MS) analysis. Numerous separation techniques have been used, including one- and two-dimensional gel electrophoresis, chromatography, and affinity techniques. MS has become a powerful method for analyzing biologic samples. This review does not cover all aspects of proteomics, but is intended to give an introductory explanation of the technology using the example of the proteomics of nasal secretions.

Biomarkers↗
PubMed2026

Enzyme kinetics shapes the growth response of metabolic networks.

Microbes adjust their metabolism to environmental challenges by changing protein expression levels, metabolite concentrations, and reaction rates. Average expression levels in large proteome sectors change coherently, while individual proteins show divergent shifts even within the same pathway. Here, we establish a metabolic model that integrates local enzyme kinetics and global network architecture to predict the joint growth response of proteins and metabolites. Under nutrient limitation, we predict a remarkably simple pattern of proteome reallocation with growth rate: protein expression levels change linearly but heterogeneously. For a given enzyme, the direction of change is determined by its local kinetic constants - catalytic rate and substrate affinity - and by the degree of nutrient restriction affecting its embedding pathway. This double-graded growth response of the proteome is mediated by restriction-dependent metabolite levels, which are predicted to decrease with growth rate in a nonlinear way. The model establishes three specific growth laws: protein expression changes of individual enzymes are negatively correlated with their expression and with their substrate saturation at high growth; average changes of pathways and larger functional sectors are correlated with their internal variance. These predictions are in quantitative agreement with measured system-wide proteomics and metabolomics data of E. coli. Enzyme-specific response patterns are a starting point for model-guided interventions into bacterial metabolism.

Kinetics↗
PubMed2007

A rapid, economical, and reproducible method for human serum delipidation and albumin and IgG removal for proteomic analysis.

Serum is a readily available source for diagnostic assays, but the identification of disease-specific serum biomarkers has been impeded by the dominance of human serum albumin (HSA) and immunoglobulin G (IgG) in the serum proteome. Therefore, in order to observe lower-abundance serum proteins, removal or depletion of at least these two proteins is required. However, the depletion method needs to be inexpensive and reproducible. We describe such a protocol that combines delipidation by centrifugation, IgG removal with Protein G Sepharose, and HSA depletion with sodium chloride/ethanol precipitation. The protocol is streamlined to increase reproducibility and is compatible with many proteomic platforms, including two-dimensional gel electrophoresis, and high-performance liquid chromatography either offline or coupled online with a mass spectrometer. The reproducible depletion of lipids, IgG, and HSA permits a higher load of the remaining serum proteins, facilitating the identification of disease biomarkers.

Biomarkers↗
PubMed2006

Different immunoaffinity fractionation strategies to characterize the human plasma proteome.

Plasma proteins may often serve as indicators of disease and are a rich source for biomarker discovery. However, the intrinsic large dynamic range of plasma proteins makes the analysis very challenging because a large number of low abundance proteins are often masked by a few high abundance proteins. The use of prefractionation methods, such as depletion of higher abundance proteins before protein profiling, can assist in the discovery and detection of less abundant proteins that may ultimately prove to be informative biomarkers. But there are few studies on comprehensive investigation of the proteins both in the fractions depleted and remainder. In the present study, two different immunoaffinity fractionation columns for the top-6 or the top-12 proteins in plasma were investigated and both the proteins in column-bound and flow-through fractions were subsequently analyzed. A two-dimensional peptide separation strategy, utilizing chromatographic separation techniques, combined with tandem mass spectrometry (MS/MS) was employed for proteomic analysis of the four fractions. Using the established HUPO PPP criteria, a total of 2401 unique plasma proteins were identified. The Multiple Affinity Removal System yielded 921 and 725 unique proteins from the flow-through and bound fractions, respectively, whereas the Seppro MIXED 12 column yielded identification of 897 and 730 unique proteins from the flow-through and bound fractions, respectively. When more stringent criteria, based on searching against the reversed database, were implemented, 529 unique proteins were identified from the four fractions with the confidence in peptide identification increased from 73.6% to 99%. To determine whether the presence of nontarget proteins in the immunoaffinity-bound fraction could be attributed to their interaction with high abundance proteins, co-immunoprecipitation analysis with an antibody to human plasma albumin was performed, which resulted in an identification of 40 unique proteins from the coimmunoprecipitate with the more stringent criteria. This study illustrated that combining the column-bound and flow-through fractions from immunoaffinity separation affords more extensive profiling of the protein content of human plasma. The presence of nontarget proteins in the column-bound fractions may be induced by their binding to the higher abundance proteins targeted by the immunoaffinity column.

Amino Acid Sequence↗
PubMed2005

Quantitative proteomic analysis of age-related changes in human cerebrospinal fluid.

Identification of cerebrospinal fluid (CSF) biomarkers of the common age-related neurodegenerative diseases would be of great value to clinicians because of the difficulties in differential diagnoses of these diseases in clinical practice. Proteins are one class of potential biomarkers currently under investigation in the hope that different ensembles of proteins will aid in the diagnosis of these diseases, as well as in the assessment of progression and response to therapy. However, before undertaking a rational approach to CSF protein biomarkers of age-related neurodegeneration, we must first systematically identify CSF proteins and determine whether their levels change with normal aging. In this study, we used a powerful shotgun proteomic method, two-dimensional microcapillary liquid chromatography electrospray ionization tandem mass spectrometry, to identify proteins in human CSF. Additionally, using pooled CSF samples, we quantitatively compared the CSF proteome of younger adults with that of older adults using isotope-coded affinity tags (ICAT). From these studies we identified more than 300 proteins in CSF and found that there were 30 proteins with >20% change in concentrations between older and younger individuals. Finally, we validated changes in concentration for two of these proteins using Western blots in CSF from a separate set of individuals. These data not only expand substantially our current knowledge regarding human CSF proteins, but also supply the necessary information to appropriately interpret protein biomarkers of age-related neurodegenerative diseases.

Adult↗
PubMed2007

Mitochondrial phosphoproteome revealed by an improved IMAC method and MS/MS/MS.

IMAC in combination with mass spectrometry is a promising approach for global analysis of protein phosphorylation. Nevertheless this approach suffers from two shortcomings: inadequate efficiency of IMAC and poor fragmentation of phosphopeptides in the mass spectrometer. Here we report optimization of the IMAC procedure using (32)P-labeled tryptic peptides and development of MS/MS/MS (MS3) for identifying phosphopeptide sequences and phosphorylation sites. The improved IMAC method allowed recovery of phosphorylated tryptic peptides up to approximately 77% with only minor retention of unphosphorylated peptides. MS3 led to efficient fragmentation of the peptide backbone in phosphopeptides for sequence assignment. Proteomics of mitochondrial phosphoproteins using the resulting IMAC protocol and MS3 revealed 84 phosphorylation sites in 62 proteins, most of which have not been reported before. These results revealed diverse phosphorylation pathways involved in the regulation of mitochondrial functions. Integration of the optimized batchwise IMAC protocol with MS3 offers a relatively simple and more efficient approach for proteomics of protein phosphorylation.

Animals↗
PubMed2006

Quantitative proteomic profiling of pancreatic cancer juice.

Pancreatic juice is an exceptionally rich source of cancer-specific proteins shed from cancerous ductal cells into the pancreatic juice. Quantitative proteomic analysis of the proteins specific to pancreatic cancer juice has not previously been reported. We used isotope-code affinity tag (ICAT) technology and MS/MS to perform quantitative protein profiling of pancreatic juice from pancreatic cancer patients and normal controls. ICAT technology coupled with MS/MS allows the systematic study of the proteome and measures the protein abundance in pancreatic juice with the potential for development of biomarkers. A total of 105 proteins were identified and quantified in the pancreatic juice from a pancreatic cancer patient, of which 30 proteins showed abundance changes of at least twofold in pancreatic cancer juice compared to normal controls. Many of these proteins have been externally validated. This is the first comprehensive study of the pancreatic juice proteome by quantitative global protein profiling, and the study reveals numerous proteins that are shown for the first time to be associated with pancreatic cancer, providing candidates for diagnostic biomarkers. One of the identified proteins, insulin-like growth factor binding protein-2 was further validated by Western blotting to be elevated in pancreatic cancer juice and overexpressed in pancreatic cancer tissue.

Amino Acid Sequence↗
PubMed2006

Population proteomics: the concept, attributes, and potential for cancer biomarker research.

This review outlines the concept of population proteomics and its implication in the discovery and validation of cancer-specific protein modulations. Population proteomics is an applied subdiscipline of proteomics engaging in the investigation of human proteins across and within populations to define and better understand protein diversity. Population proteomics focuses on interrogation of specific proteins from large number of individuals, utilizing top-down, targeted affinity mass spectrometry approaches to probe protein modifications. Deglycosylation, sequence truncations, side-chain residue modifications, and other modifications have been reported for myriad of proteins, yet little is know about their incidence rate in the general population. Such information can be gathered via population proteomics and would greatly aid the biomarker discovery efforts. Discovery of novel protein modifications is also expected from such large scale population proteomics, expanding the protein knowledge database. In regard to cancer protein biomarkers, their validation via population proteomics-based approaches is advantageous as mass spectrometry detection is used both in the discovery and validation process, which is essential for the detection of those structurally modified protein biomarkers.

Biomarkers, Tumor↗
PubMed2005

On-line concentration of peptides and proteins with the hyphenation of polymer monolithic immobilized metal affinity chromatography and capillary electrophoresis.

An iminodiacetic acid (IDA)-type adsorbent is prepared at the one end of a capillary by covalently bonding IDA to the monolithic rods of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate). Cu(II) is later introduced to the support via the interaction with IDA. By this means, polymer monolithic immobilized metal affinity chromatography (IMAC) materials are prepared. With such a column, IMAC for on-line concentration and capillary electrophoresis (CE) for the subsequent analysis are hyphenated for the analysis of peptides and proteins. The reproducibility of such a column has been proved good with relative standard deviations (RSDs) of dead time of less than 5% for injection-to-injection and 12% for column-to-column (n = 3). Through application on the analysis of standard peptides and real protein samples, such a technique has shown promising in proteome study.

Chromatography, Affinity↗
PubMed2005

Proteomic insights into ubiquitin and ubiquitin-like proteins.

The dynamic and specific modification of cellular proteins by members of the ubiquitin protein family is a vital regulatory mechanism that lies at the heart of almost all biological processes. Because of both their pervasive and complex nature, these regulatory pathways have been the target of many recent proteomic studies. Such works have provided numerous insights. Through the use of various mass spectrometry techniques, affinity purification methods, and/or chemical probes, large lists have begun to be compiled for the multitude of substrates, interacting partners, and enzymatic components of these regulatory circuits. Furthermore, similar tools have provided many insights into functional aspects such as their mechanisms of substrate specificity and enzymatic activity. This review provides a summary of these recent proteomic works, along with comments on future directions of the field.

Humans↗
PubMed2001

Proteomics: posttranslational modifications, immune responses and current analytical tools.

The publication of the human genome sequence enables most of the still unknown protein sequences to be added to the current databases. A sequence alone does not, however, give information about the possible expression level of the corresponding protein, neither does it inform about the possible posttranslational modifications, like phosphorylation, glycosylation or changes in individual amino acids. Thus, the human proteome project, a large scale analysis of the functions of gene products, will have an enormous impact on our understanding of the biochemistry of proteins, processes and pathways they are involved in. The diversity in proteins is considerably expanded by various posttranslational modifications. These also pose problems to the investigators, but their careful analysis often pays back because they can reveal important properties in proteins or peptides--like an increased antigenicity leading to (auto)immune responses or an active form of a signaling protein. Immune tolerance usually exists towards self-proteins, but in specific cases it may be broken by posttranslational modifications in the proteins. Novel mass spectrometric, affinity and display techniques offer valuable tools for the large-scale analysis of proteomes. In the present paper we discuss their use for the detection of posttranslational modifications, functional interactions and possible disease-associated abnormalities in proteins.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2005

Characterisation of kinase-selective inhibitors by chemical proteomics.

Low-molecular-weight inhibitors of protein kinases are extensively used as research tools in signal transduction analysis and constitute a rapidly growing class of therapeutics for targeted intervention in human diseases. To determine how kinase-selective drugs interfere with cellular physiology on the molecular level, experimental strategies relying on the affinity capture of cellular targets in combination with protein identification by mass spectrometry have been established for a variety of kinase inhibitors. Importantly, these chemical proteomic methods permit the direct analysis of kinase inhibitor selectivity in biological systems and have led to new insights into the cellular modes of action of kinase-selective small molecule antagonists.

Animals↗
PubMed2004

A proteomic approach to identify phosphoproteins encoded by cDNA libraries.

We report a method for large-scale rapid analysis of phosphoproteins in tissues or cells by combining immobilized metal affinity chromatography (IMAC) with phage display cDNA library screening. We expressed a testis cDNA library as fusion proteins on phage and, using IMAC, enriched for sequences encoding phosphoproteins. Selected clones were polymerase chain reaction amplified and sequenced. The majority of the clones sequenced (80%) encoded known proteins previously identified as phosphoproteins. Immunoblotting with phosphotyrosine antibodies confirmed that some of the selected sequences encoded tyrosine phosphorylated proteins when expressed on phage. An advantage of this method is the rapid identification of phosphoproteins encoded by a cDNA library, which can identify proteins that are potentially phosphorylated in vivo. When this method is combined with limited enzymatic digestion and tandem mass spectrometric techniques, the specific phosphorylation site in a protein can be identified. This technique can be used in proteomics studies to effectively detect phosphorylated proteins and avoid time-consuming and expensive peptide sequencing.

Chromatography, Affinity↗
PubMed2006

Proteomic analysis of high-density lipoprotein.

Plasma lipoproteins, such as high-density lipoprotein (HDL), can serve as carriers for a wide range of proteins that are involved in processes such as lipid metabolism, thrombosis, inflammation and atherosclerosis. The identification of HDL-associated proteins is essential with regards to understanding these processes at the molecular level. In this study, a combination of proteomic approaches including 1-DE and 2-DE MALDI-TOF, isotope-coded affinity tag and Western blot analysis were employed to identify proteins associated with human HDL. To minimize potential losses of HDL-associated proteins during isolation, a one-step ultracentrifugation technique was applied and the quality of purified HDL was confirmed by nephelometry, high-performance gel chromatography, and Western blot analysis. MS analysis revealed the presence of 56 HDL-associated proteins including all known apolipoproteins and lipid transport proteins. Furthermore, proteins involved in hemostasis and thrombosis, the immune and complement system were found. In addition, growth factors, receptors, hormone-associated proteins and many other proteins were found to be associated with HDL. Our approach thus resulted in the identification of a large number of proteins associated with HDL. The combination of proteomic technologies proved to be a powerful and comprehensive tool for the identification of proteins on HDL.

Blood Proteins↗
PubMed2003

Quantitative proteome analysis: methods and applications.

With the completion of the genomic sequencing of a number of species, including that of humans, much attention is currently focused on how the information in these sequences might be interpreted in terms of the structure, function, and control of biologic systems and processes. Quantitative proteome analysis, the global analysis of protein expression, is increasingly being used as a method to study steady-state and perturbation-induced changes in protein profiles. The rationale for quantitative proteome analysis is described, along with a new technology for high throughput quantitative profiling of proteins in complex mixtures and its current status with selected applications.

Affinity Labels↗
PubMed2002

An efficient protein complex purification method for functional proteomics in higher eukaryotes.

The ensemble of expressed proteins in a given cell is organized in multiprotein complexes. The identification of the individual components of these complexes is essential for their functional characterization. The introduction of the 'tandem affinity purification' (TAP) methodology substantially improved the purification and systematic genome-wide characterization of protein complexes in yeast. The use of this approach in higher eukaryotic cells has lagged behind its use in yeast because the tagged proteins are normally expressed in the presence of the untagged endogenous version, which may compete for incorporation into multiprotein complexes. Here we describe a strategy in which the TAP approach is combined with double-stranded RNA interference (RNAi) to avoid competition from corresponding endogenous proteins while isolating and characterizing protein complexes from higher eukaryotic cells. This strategy allows the determination of the functionality of the tagged protein and increases the specificity and the efficiency of the purification.

Animals↗
PubMedDate not supplied

[Clinical proteomics: towards early detection of cancers].

A key challenge in clinical proteomic of cancer is the identification of biomarkers that would allow early detection, diagnosis and monitor progression of the disease to improve long-term survival of patients. Recent advances in proteomic instrumentation and computational methodologies offer unique chance to rapidly identify these new candidate markers or pattern of markers. The combination of retentate affinity chromatography and surfaced-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry is one of the most interesting new approaches for cancer diagnostic using proteomic profiling. This review aims to summarize the results of studies that have used this new technology method for the early diagnosis of human cancer. Despite promising results, the use of the proteomic profiling as a diagnostic tool brought some controversies and technical problems and still requires some efforts to be standardised and validated.

Biomarkers, Tumor↗