Two-dimensional liquid separations–mass mapping of proteins from human cancer cell lysates

Lubman, David M.; Kachman, Maureen; Wang, Haixing; Gong, Siyuan; Yan, Fang; Hamler, Rick; O'neil, Kimberly A.; Zhu, K.; Buchanan, Nathan S.; Barder, Timothy J.

doi:10.1016/s1570-0232(02)00551-2

Cited by 113 publications

(89 citation statements)

References 52 publications

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“…A saturated CHCA in 50% acetonitrile and 1% TFA was diluted to a 1:4 ratio (v/v) in 50% acetonitrile and 1% TFA. Angiotensin I, ACTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), and ACTH (18-39) were added to the 1:4 diluted matrix solution. The resulting matrix-standard solution was vortexed.…”

Section: Maldi Sample Preparation-mentioning

confidence: 99%

“…Each band represents a protein peak eluted from the RP-HPLC separation with gray scale intensity representing the relative intensity of each protein peak. The image offers the same advantages of differential expression profiling provided by a 2-D gel analysis but is obtained in digitized form [12]. The CF step in this experiment assayed 17 pI fractions ranging from 4.0-7.4.…”

Section: -D Liquid Separationmentioning

confidence: 99%

“…The method is based upon pI separation in the first dimension and non-porous RP-HPLC in the second dimension. The eluant of the HPLC can be directly introduced into electrospray-time of flight mass spectroscopy (ESI-TOF MS) for analysis of the intact protein M r [12]. The M r together with the pI can unambiguously identify complex proteins.…”

Section: Introductionmentioning

confidence: 99%

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Identification of metastasis‐associated proteins in a human tumor metastasis model using the mass‐mapping technique

et al. 2004

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For most cancer cell types, the acquisition of metastatic ability leads to clinically incurable disease. The identification of molecules whose expression is specifically correlated with the metastatic spread of cancer would facilitate the design of therapeutic interventions to inhibit this lethal process. In order to facilitate metastasis gene discovery we have previously characterized a pair of monoclonal cell lines from the human breast carcinoma cell line MDA-MB-435 that have different metastatic phenotypes in immune-compromised mice. In this study, serum-free conditioned media was collected from the cultured monoclonal cell lines and a mass mapping technique was applied in order to profile a component of each cell line proteome. We utilized chromatofocusing in the first dimension to obtain a high resolution separation based on protein pI, and nonporous silica reverse-phase high performance liquid chromatography was used for the second dimension. Selected proteins were identified on the basis of electrospray ionization time of flight mass spectrometry (ESI-TOF MS) intact protein mapping and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting. Using this approach we were able to map over 400 proteins and plot them as a 2-D map of pI versus accurate M r . This was performed over a pI range of 4.0-6.2, and a mass range of 6-80 kDa. ESI-TOF MS data and further analysis using MALDI-TOF MS confirmed and identified 27 differentially expressed proteins. Proteins associated with the metastatic phenotype included osteopontin and extracellular matrix protein 1, whereas the matrix metalloproteinase-1 and annexin 1 proteins were associated with the non-metastatic phenotype. These findings demonstrate that the mass mapping technique is a powerful tool for the detection and identification of proteins in complex biological samples and which are specifically associated with a cellular phenotype.

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Section: Maldi Sample Preparation-mentioning

confidence: 99%

Section: -D Liquid Separationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Identification of metastasis‐associated proteins in a human tumor metastasis model using the mass‐mapping technique

et al. 2004

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“…Both the SEC/IEX-RPLC systems have also been successfully interfaced with electrospray TOF-MS (ESI-MS). The quantitative proteomic profiling of proteins using multidimensional chromatography has also been performed (reviewed in [20]). A number of proteomic studies have been performed utilising such techniques [21][22][23].…”

Section: Introductionmentioning

confidence: 99%

Multidimensional liquid phase protein separations in conjunction with stable isotope labelling for quantitative proteomics

et al. 2007

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A method for quantitative protein profiling has been developed utilising multidimensional liquid phase protein separations in conjunction with stable isotope labelling. This approach combines the advantages of high throughput, automated, reproducible protein separations with accurate protein quantitation performed in the mass spectrometer. Escherichia coli cells were grown in the presence and absence of the DNA methylation inhibitor 5-Azacytidine on 14 N and 15 N enriched media. Protein separations were performed using ion exchange chromatography in the first dimension and RP capillary chromatography in the second dimension. UV absorbance measurements were used for the initial semiquantitative identification of differentially expressed proteins. Selected peaks from the mixed 15 N/ 14 N lysates were used for the accurate quantitation performed in the mass spectrometer using the ratios of the stable isotopes. Using this approach, a number of differentially expressed proteins have been identified. Moreover, this approach overcomes a number of caveats associated with multidimensional liquid phase protein separations, including the presence of multiple proteins present in a single chromatographic peak.

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“…Similarly, a novel 3-dimension liquid chromatography strategy by coupling of hydrophobic interaction chromatography (HIC) and reverse phase chromatography with top-down MS separated and identified a total of 640 proteins from HEK 293 cell lysate protein fractions [36]. On the other hand, chromatofocusing and solution isoelectric focusing (both utilizes pH gradient) are able to separate proteins with high isoelectric point correlation and are considered as alternative methods to salt gradient ion exchange chromatography for top-down proteomic separations [35,37]. Strong anion exchange coupled to RPLC-MS for the separation of intact proteins has also been reported for the study of E. Coli [38].…”

Section: Reverse Phase Liquid Chromatography [Rplc]mentioning

confidence: 99%

Top-down proteomics: applications, recent developments and perspectives

Pamreddy

Panyala

2016

J Appl Bioanal

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REVIEWNow-a-days, top-down proteomics (TDP) is a booming approach for the analysis of intact proteins and it is attaining significant interest in the field of protein biology. The term has emerged as an alternative to the well-established, bottom-up strategies for analysis of peptide fragments derived from either enzymatically or chemically digestion of intact proteins. TDP is applied to mass spectrometric analysis of intact large biomolecules that are constituents of protein complexes and assemblies. This article delivers an overview of the methodologies in top-down mass spectrometry, mass spectrometry instrumentation and an extensive review of applications covering the venomics, biomedical research, protein biology including the analysis of protein post-translational modifications (PTMs), protein biophysics, and protein complexes. In addition, limitations of top-down proteomics, challenges and future directions of TDP are also discussed.

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Two-dimensional liquid separations–mass mapping of proteins from human cancer cell lysates

Cited by 113 publications

References 52 publications

Identification of metastasis‐associated proteins in a human tumor metastasis model using the mass‐mapping technique

Identification of metastasis‐associated proteins in a human tumor metastasis model using the mass‐mapping technique

Multidimensional liquid phase protein separations in conjunction with stable isotope labelling for quantitative proteomics

Top-down proteomics: applications, recent developments and perspectives

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