Protein microarrays: Meeting analytical challenges for clinical applications

Liotta, Lance A.; Espina, Virginia; Mehta, Arpita; Calvert, Valerie; Rosenblatt, Kevin P.; Geho, David; Munson, Peter J.; Young, Lynn; Wulfkuhle, Julia; Petricoin, Emanuel F.

doi:10.1016/s1535-6108(03)00086-2

Cited by 425 publications

(330 citation statements)

References 52 publications

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“…The reverse phase protein microarray construction was previously reported [13,14]. Briefly, samples were lysed, loaded into 384-well plates and printed in Total protein values were assessed by staining the slides with Sypro Ruby Blot Stain (Molecular Probes, Eugene, OR).…”

Section: Reverse Phase Protein Microarray Constructionmentioning

confidence: 99%

Kinase activation profile associated with TGF-β-dependent migration of HCC cells: a preclinical study

Fransvea

Mazzocca

Santamato

et al. 2010

Cancer Chemother Pharmacol

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2 AbstractPurpose. To identify the molecular mechanisms responsible for tumor cell migration is essential for developing agents that can prevent the relapse or the metastatic spread of hepatocellular carcinoma (HCC). Methods. In this study, we investigated the effects of the transforming growth factor-receptor I inhibitor LY2109761 on two different human HCC cell lines, in vitro and in vivo. Results.LY2109761 inhibits HCC migration in a dose-dependent manner. This inhibition is associated with decreased phosphorylation of SMAD-2, FAK and 1-integrin, and with increased levels of E-cadherin. By contrast, LY2109761 did not alter the phosphorylation pattern of p38MAPkinase. In a two-and a three-day time-course and in dose-titration experiments, LY2109761 inhibited HCC migration as well as phospho-SMAD-2 and the adhesion proteins. LY2109761 showed the best effect on day 2 at 1 nM and for three days at 100 nM concentration. This suggests that maximum effects were sustained for several days and were not dependent on excess concentrations. Finally, in a xenograft model of HCC, LY2109761 strongly inhibits tumor growth, intravasation and metastasis at the aforementioned lower concentrations. Conclusions. In conclusion, inhibition of transforming growth factor-TGF-) appears to occur at low concentrations of LY2109761, that displays multiple effects on kinases that control HCC cell migration. These findings may help the design of future clinical trials with inhibitors of TGF-.

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Section: Reverse Phase Protein Microarray Constructionmentioning

confidence: 99%

Kinase activation profile associated with TGF-β-dependent migration of HCC cells: a preclinical study

Fransvea

Mazzocca

Santamato

et al. 2010

Cancer Chemother Pharmacol

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“…In contrast to forward-phase arrays, in reverse-phase arrays multiple analytes are immobilized and probed with specific antibodies [36,37] (Figure 2). With RPAs, cellular lysates are spotted directly onto the array surface.…”

Section: Reverse-phase Protein Microarraysmentioning

confidence: 99%

“…Arraying each lysate in a dilution curve series is critical for ensuring that at least part of the assay can be assessed within the linear range of a particular antibody-antigen reaction and also allows for more accurate quantification. The technique has been shown to be robust in terms of intra-and inter-array variation (CV less than 10%), and with a sensitivity of less than 50 fg/µl [36,37,39]. The latter depends significantly on the affinity of the probing antibody.…”

Section: Reverse-phase Protein Microarraysmentioning

confidence: 99%

Array-based proteomics: mapping of protein circuitries for diagnostics, prognostics, and therapy guidance in cancer

et al. 2006

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The human proteome, due to the enormity of post-translational permutations that result in large numbers of isoforms, is much more complex than the genome and alterations in cancer can occur in ways that are not predictable by translational analysis alone. Proteomic analysis therefore represents a more direct way of investigating disease at the individual patient level. Furthermore, since most novel therapeutic targets are proteins, proteomic analysis potentially has a central role in patient care. At the same time, it is becoming clear that mapping entire networks rather than individual markers may be necessary for robust diagnostics as well as tailoring of therapy. Consequently, there is a need for high-throughput multiplexed proteomic techniques, with the capability of scanning multiple cases and analysing large numbers of endpoints. New types of protein arrays combined with advanced bioinformatics are currently being used to identify molecular signatures of individual tumours based on protein pathways and signalling cascades. It is envisaged that analysing the cellular 'circuitry' of ongoing molecular networks will become a powerful clinical tool in patient management.

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“…This method assesses only one protein per microarray, but it nonetheless has a great advantage because multiple samples can be analyzed and compared side by side in a single array [13,14].…”

Section: Introductionmentioning

confidence: 99%

“…Using the HRP-DAB platform, detection of proteins in single cell can be accomplished routinely [13]. However, the HRP-DAB system is sensitive to various factors, such as temperature, reagent quality, and specific activity of the HRP enzyme.…”

Section: Introductionmentioning

confidence: 99%

Quantum dots-based reverse phase protein microarray

Shingyoji

Gerion

Pinkel

et al. 2005

Talanta

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CdSe nanocrystals, also called quantum dots (Qdots) are a novel class of fluorophores, which have a diameter of a few nanometers and possess high quantum yield, tunable emission wavelength and photostability. They are an attractive alternative to conventional fluorescent dyes. Quantum dots can be silanized to be soluble in aqueous solution under biological conditions, and thus be used in bio-detection. In this study, we established a novel Qdot-based technology platform that can perform accurate and reproducible quantification of protein concentration in a crude cell lysate background. Protein lysates have been spiked with a target protein, and a dilution series of the cell lysate with a dynamic range of three orders of magnitude has been used for this proof-of-concept study. The dilution series has been spotted in microarray format, and protein detection has been achieved with a sensitivity that is at least comparable to standard commercial assays, which are based on horseradish peroxidase (HRP) catalyzed diaminobenzidine (DAB) chromogenesis. The data obtained through the Qdot method has shown a close linear correlation between relative fluorescence unit and relative protein concentration. The Qdot results are in almost complete agreement with data we obtained with the well-established HRP-DAB colorimetric array (R 2 =0.986). This suggests that Qdots can be used for protein quantification in microarray format, using the platform presented here.

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Protein microarrays: Meeting analytical challenges for clinical applications

Cited by 425 publications

References 52 publications

Kinase activation profile associated with TGF-β-dependent migration of HCC cells: a preclinical study

Kinase activation profile associated with TGF-β-dependent migration of HCC cells: a preclinical study

Array-based proteomics: mapping of protein circuitries for diagnostics, prognostics, and therapy guidance in cancer

Quantum dots-based reverse phase protein microarray

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