Isoelectric Focusing Nonporous RP HPLC:  A Two-Dimensional Liquid-Phase Separation Method for Mapping of Cellular Proteins with Identification Using MALDI-TOF Mass Spectrometry

Wall, Daniel; Kachman, Maureen; Gong, Siyuan; Hinderer, Robert; Parus, Stephen J.; Misek, David E.; Hanash, Samir M.; Lubman, David M.

doi:10.1021/ac991332t

Cited by 235 publications

(152 citation statements)

References 44 publications

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“…MS separates peptides or proteins according to their mass-to-charge ratio, and can be used to screen for differential expression directly, or by coupling to upstream protein separation methods [16]. MALDI-TOF-MS analysis is now a routine tool for the identification of proteins separated by 2D methods [17]. MALDI-TOF has also been applied directly to identify peptides sequestered from the serum of breast cancer patients.…”

Section: Proteomic Profiling Of Human Tissues and Fluidsmentioning

confidence: 99%

Breast tumor metastasis: analysis via proteomic profiling

Goodison¹,

Urquidi²

2008

Expert Review of Proteomics

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The ability to predict the metastatic behavior of a patient's cancer, as well as to detect and eradicate such recurrences, remain major clinical challenges in oncology. While many potential molecular biomarkers have been identified and tested previously, none have greatly improved the accuracy of specimen evaluation over routine histopathological criteria and, to date, they predict individual outcomes poorly. The ongoing development of high-throughput proteomic profiling technologies is opening new avenues for the investigation of cancer and, through application in tissue-based studies and animal models, will facilitate the identification of molecular signatures that are associated with breast tumor cell phenotype. The appropriate use of these approaches has the potential to provide efficient biomarkers, and to improve our knowledge of tumor biology. This, in turn, will enable the development of targeted therapeutics aimed at ameliorating the lethal dissemination of breast cancer. In this review, we focus on the accumulating proteomic signatures of breast tumor progression, particularly those that correlate with the occurrence of distant metastases, and discuss some of the expected future developments in the field.

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Section: Proteomic Profiling Of Human Tissues and Fluidsmentioning

confidence: 99%

Breast tumor metastasis: analysis via proteomic profiling

Goodison¹,

Urquidi²

2008

Expert Review of Proteomics

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“…Elution of bound hydrophobic protein is facilitated by enhancing the concentration of organic solvent, thereby increasing the retention time. Reverse phase chromatography as cited in [55,57,58] is usually done alongside mass spectrometry in a way to analyze the protein that is eluted from the column [50].Wall et al [51] described a two-dimensional liquid-phase separation method coupled with a MALDI-TOF mass spectrometer to separate and profile the cellular proteins, by using isoelectric focusing in the first dimension and RP-HPLC in the second dimension and the selected fractions were analyzed using MALDI-TOF MS. By using this technique, the authors were able to resolve 700 protein bands with a high resolution and substantiated this approach as an alternative to 2-D gel separation technique.…”

Section: Reversedmentioning

confidence: 99%

Musculoskeletal ProteinAnalysis Techniques - A Review

Kamineni¹,

Manepally²,

Kamineni³

2016

JRAD

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There have been a large number of experimental methods for purifying and analyzing proteins from the sample of interest. Determination of protein concentration is often the key step and is common to many applications in protein research and life sciences. The most commonly used quantitative techniques to measure the protein concentration are Amino acid analysis, Biuret assay, Bradford assays, Folin-Lowry assay, Bicinchoninic (BCA) assays, UV absorbance assays and Antibody-based assays such as ELISA and Western Blotting. Some of the qualitative methods that are used to detect different types of proteins and amino acids are Ninhydrin test, Xanthoproteic test, Millon's test, Sulfur test, Hopkins-Cole test, and Nitroprusside test. Chromatographic analysis can also be carried out on an either qualitative or quantitative basis to purify complex protein mixtures based on their properties such as size, solubility, charge, hydrophobicity and bio-specific interaction. Selection of these assays is based upon the ease of performance, range of concentrations, sensitivity, and interfering substances contained in the complex mixture. The purpose of this review is to provide an assessment of commonly used chromatographic and colorimetric methods for the determination of protein concentration.

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“…A two-dimensional all liquid-phase method combined with MS for protein profile analysis was developed. Proteins are first fractionated by pI using IEF and then further separated according to hydrophobicity by reverse-phase HPLC (74). To improve its quantitative and high throughput capacity and its reproducibility, LC may be coupled to sample isotope labeling as in the ICAT technique (75).…”

Section: Mass Spectrometry-based Approachesmentioning

confidence: 99%

Proteomics of Breast Cancer

Bertucci

Birnbaum

Gonçalvès

2006

Molecular & Cellular Proteomics

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Progresses in screening, early diagnosis, prediction of aggressiveness and of therapeutic response or toxicity, and identification of new targets for therapeutic will improve survival of breast cancer. These progresses will likely be accelerated by the new proteomic techniques. In this review, we describe the different techniques currently applied to clinical samples of breast cancer and the most important results obtained with the two most popular proteomic approaches in translational research (tissue microarrays and SELDI-TOF). Molecular & Cellular Proteomics 5:1772-1786, 2006.Worldwide, breast cancer is one of the most frequent and deadly cancers. Although the survival of patients has increased over the last decades in relation with screening programs and postoperative adjuvant systemic therapies (hormone therapy and chemotherapy), many patients die from metastatic relapse. Breast cancer is a complex disease. Accumulation of numerous and often unknown molecular alterations causes cell proliferation, genetic instability, and acquisition of an increasingly invasive and resistant phenotype. The combinatorial origin and the heterogeneity of malignant cells and the variability of the host background create molecularly distinct subgroups of tumors endowed with different phenotypes and clinical outcomes. This heterogeneity is only partially apprehended by the clinicopathological parameters currently used by clinicians, hampering the selection of the most appropriate diagnostic or therapeutic strategy for each case. In addition, current cytotoxic drugs do not differentiate between cancerous and normal cells, causing sometimes disastrous adverse effects. Major efforts aim at characterizing the heterogeneity of the disease by defining more reliable diagnostic/prognostic factors and at developing molecular therapies selectively targeting the tumor cells.For some decades, the study of molecular alterations has successfully elucidated some mechanisms of mammary oncogenesis and identified key genes such as ERBB2, TP53, CCND1, BRCA1, and BRCA2. It has also allowed considerable therapeutic progress by targeting hormonal receptors and ERBB2/HER2 receptor. Today high throughput molecular typing provides an unprecedented opportunity to tackle the combinatory aspect and the complexity of breast cancer. A combination of markers (molecular signature) will probably be more sensitive and specific than a single molecular marker to reflect the actual heterogeneity of disease; more reliable for screening, diagnosis, prognosis, and prediction of therapeutic response; and more useful to find new therapeutic targets. The first large scale techniques applied to the cancer field were DNA microarrays for mRNA expression profiling (1). Several studies have demonstrated the clinical potential of this approach, notably by identifying new biologically relevant and prognostic subclasses of breast cancer unidentifiable by conventional means (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). With the recent development of similar technologies at the DNA...

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Isoelectric Focusing Nonporous RP HPLC: A Two-Dimensional Liquid-Phase Separation Method for Mapping of Cellular Proteins with Identification Using MALDI-TOF Mass Spectrometry

Cited by 235 publications

References 44 publications

Breast tumor metastasis: analysis via proteomic profiling

Breast tumor metastasis: analysis via proteomic profiling

Musculoskeletal ProteinAnalysis Techniques - A Review

Proteomics of Breast Cancer

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