Protein profiling pdf

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Log in with Facebook Log in with Google. Remember me on this computer. Enter the email address you signed up with and we'll email you a reset link. Need an account? Click here to sign up. Download Free PDF. Paul Tam. A short summary of this paper. Tel: , Fax: , E-mail: qyhe hku.

By comparing with normal control, a number of protein or polypeptide signals were found significantly and consistently different in their intensities expression levels in tumor sera. Interestingly, 9 polypeptide peaks in these proteomic features can be simultaneously detected with consistent variations by more than one type of protein chips.

None of the expression differences of these 9 polypeptides was found in similar comparisons between healthy controls and hepatomas. Preliminary protein identification showed hints for that some of these proteomic alterations may be closely related to the tumorigenesis of neuroblastoma. Neuroblastoma arises from neuroectodermal cells derived from the neural crest and takes place in the adrenal medulla or paraspinal symphatetic nervous system [Schwab et al.

In general, infants less than one year old with neuroblastoma present with lower stage and less aggressive disease, which are usually chemosensitive and thus curable [Bown, ]. However, in children over one year old, the disease is often much more aggressive, metastatic and chemoresistant, and therefore has a high mortality rate [Bown, ].

The biological mechanism underlying this substantial clinical heterogeneity of neuroblastoma is still elusive. To improve the understanding of neuroblastoma, many genetic and biological features have been investigated [Riley et al. A number of studies have also been performed to identify tumor markers for facilitating the screening, diagnosis, prognosis, or monitoring of the disease to improve cure rates [Riley et al.

However, specific and sensitive diagnostic and prognostic markers have not been found yet. A recent study also revealed that relapse or progression in neuroblastoma cannot be reliably detected by monitoring the current tumor markers alone [Simon et al. This presents a great need of continued effort to identify useful biomarkers for designing novel diagnostic tools and therapeutic strategies.

As an emerging powerful biological research technique, proteomics has recently become a key tool in the identification of biomarkers and disease targets [He et al.

An advantage of proteomics is its ability to simultaneously inspect the whole proteome so that correlated proteins altered in expression and modification corresponding to a disease condition can be identified in a single experiment. However, 2D-gel electrophoresis has its limitation in protein separation. For example, proteins that have molecular weights MW less than 10 kDa and isoelectric points pI less than 3 and higher than 11 cannot be separated or well displayed in gels.

In this study, serum specimens from neorublastoma patients were analyzed by using five types of protein chips. By comparing the serological protein profiles between patients and healthy controls, a number of proteomic features were found significantly altered.

Some of the alterations can be detected in more than one type of protein chips simultaneously. None of these expression differences was found in similar comparisons between normal samples and hepatomas. The study was approved by the research ethics committee of the University of Hong Kong.

Since few neuroblastoma cases were available, we only included 5 serum samples from patients admitted in Queen Mary Hospital in last two years.

Serum samples from 10 healthy subjects were used as normal control. For comparison, another sample group containing 8 normal and 8 hepatoma sera was recruited and subjected to similar screening. These protein chips have different surface affinity properties with IMAC3 for metal- binding proteins, WCX2 weak cation exchanger for positive-charge molecules, SAX2 strong anion exchanger for negative charge molecules, H50 for hydrophobic peptides and NP20 normal phase for general binding of proteins.

Each serum fraction was thawed and pretreated before loading. After vortexing, each sample was diluted in with corresponding binding buffers. This was repeated twice. The chips were then incubated at room temperature for 30 min on a shaker. After incubation, non-bound proteins and other contaminants were removed by washing with buffers for three times. These buffers were 0. Finally all chips were washed with MilliQ-deionized water for four times to remove interfering substances such as salts and detergents.

On drying, 0. The detector was run at a sensitivity of 9. Spectral analysis was carried out using the ProteinChip software version 3. All mass spectra were normalized to have the same total ion current. Cluster analysis of the detected signals and the determination of respective p values were carried out with the Biomarker Wizard Program Version 3.

This volume focuses on explorative activity-based proteomics, biomedical applications of activity-based proteomics, and chemical strategies in activity-based proteomics providing a concise overview of activity-based protein profiling.

Proteomics research requires methods to characterize the expression and function of proteins in complex mixtures.

Skip to content. Geurink, Laurette M. Prely, Gijs A. Heal und Edward W. Overkleeft Publisher: Humana Press ISBN: Category: Science Page: View: This volume focuses on explorative activity-based proteomics, biomedical applications of activity-based proteomics, and chemical strategies in activity-based proteomics providing a concise overview of activity-based protein profiling. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

A peptide mass list was generated for and , at high resolution revealed that after 6 h of the subsequent database search. For semi-quantitative labeling a considerable isotopomer peak was already labeling measurements, the area of the M15 peak was present Fig. A SD was obtained to 24 h of labeling. Significance of differences be- From the 27 excised proteins, 17 were identified which tween ratios was determined by analysis of variance.

A semi-quantitative value with respect to labeling efficiency was assigned to the proteins 2. Of the 17 proteins, two already The peptide mass list was searched with ProteinLynx showed labeling after 6 h and an additional 9 proteins Global Server Waters or the Mascot search engine were labeled after 24 h.

Proteomics , 4, — unlabeled was observed after 6 h of labeling. In order to compare our labeling procedure with an established labeling method, 3T3-L1 adipocytes were incubated with 14C -phenylalanine for 6 h. As can be seen in Fig. Collagen type 1 alpha 2 COL1A2 was present as a weaker band which can be explained by its lower abundance in our protein sample and by the fact that it has a lower labeling rate according to our measurements.

Some other bands appeared after radioactive labeling, which correspond to the analysed prominent bands on the CBB-stained gel. These bands are likely com- posed of multiple proteins making the radioactive labeling of bands different from that of single proteins as deter- mined by our MALDI-TOF approach. Labeling of individual proteins was assessed by random selection of 28 spots, which were cut from the gel and subjected to MALDI-TOF analysis for identification.

In this way, 16 proteins were identified Table 2 , of which 15 had at least one phenylalanine-containing peptide in the Figure 1. A CBB stained band pattern of proteins iso- spectrum. Of isotope label L- ring-2,3,4,5,6 2H5 phenylalanine. The the eight additional proteins, four were found to have position of the 17 bands whose corresponding proteins acquired label after 24 h as detected by isotopomer were identified are indicated see Table 1.

These proteins analysis. Two proteins, vimentin and gamma-actin, were had at least one phenylalanine containing peptide in the detected more than once. Band numbers refer to Table 1 and Fig. When labeling data of the same proteins obtained from different experiments are compared Tables 1 and 2 , some spreading is observed in the peak protein sample there is indeed a considerable difference area ratio.

As expected, the spreading is larger for pro- in labeling rates between individual proteins. For teins of which the compared isotopomer peaks are less instance, for COL1A1 a peak area ratio of 0.

One should also be aware that information about the relative labeling rate only applies to the isolated protein fraction and is a priori not a measure for the cel- lular turnover rate of proteins. Advan- tages of this method are that it is simple, nonhazardous and can easily be combined with rapid- to high-through- put standard protein profiling methods. Figure 3. COL1A1 coverage map. Underlined are tryptic Mature nondividing 3T3-L1 adipocytes were used to peptides represented as peaks in the mass spectrum.

With the Two peptides corresponding to prominent peaks of the present experimental design we were able to distinguish mass spectrum and containing phenylalanine are high- three categories of proteins in the isolated protein fraction lighted.

Indeed, a high metabolic activity of the extracellular matrix has been reported for 4 Discussion adipocytes [12—15] substantiating the discriminative power of our approach. In order to try and get an indication about rate in comparison to that of other proteins is easily the relative production rate of both proteins, these num- obtained. As such, this method may help to discriminate bers were corrected for relative amount of protein as between alternative explanations for differential protein determined by densitometry and for Mr.

This resulted in a expression. A limitation of the procedure is that this infor- replacement ratio of 2. Proteomics , 4, — Figure 4. The graphs illustrate the increasing abundance of the M15 ion due to L- ring-2,3,4,5,6 2H5 phenylala- nine incorporation after 6 and 24 h of labeling.

This stresses fiber, i. It suggests that production of the two sub- that extracellular matrix remodelling is a functional char- units occurs in mature adipocytes in a coordinated man- acteristic of mature adipocytes, at least in vitro.

At the ner. It is achieved by the enzyme collagen prolyl 4-hydroxylase which catalyses the forma- tion of 4-hydroxyproline. This enzyme is a tetramer of two different subunits, one of which is protein disulfide isom- erase band 8, Table 1; spot 12, Table 2. Since its activity probably depends on thioredoxin domains, peroxi- redoxin-1 spot 10, Table 2 might be involved in this pro- cess as well.