Two-Dimensional SEC/RPLC Coupled to Mass Spectrometry for the Analysis of Peptides

and, Gregory J. Opiteck; Jorgenson, James W.; Anderegg, Robert J.

doi:10.1021/ac961156d

Cited by 298 publications

(225 citation statements)

References 44 publications

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“…A number of systems have been developed utilising multidimensional chromatography, including the coupling of ion exchange (IEX) (first dimension) with RPLC (second dimension) [15]. In addition, SEC has been coupled to RPLC systems [16,17]. The application of parallel separations in 2-D chromatography has also been developed [18,19].…”

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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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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“…The fractions selected from RP-HPLC column were then resolved by MALDI-TOF/MS or ESI/MS. Also in another study, Opiteck et al [61] applied 2-D SEC/RPLC method coupled to electrospray-MS to analyze a mixture of peptide fragments resulting from the digestion of a protein. Owen et al [62] compared size exclusion high-performance liquid chromatography (SEC-HPLC) with an automated Immune Turbidimetric Assay (ITA) to evaluate the amount of micro albuminuria (urinary protein).…”

Section: Sizes-exclusion Chromatography (Sec)mentioning

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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“…The technique has found application in various fields, including in the separation of complex biological samples [132], polymers [133][134][135][136], natural products [130,137,138], peptides [139,140], biomolecules [141], proteins [142], food [143], wine [144,145] etc. In this section, a brief overview of some application of LC×LC for phenolic analysis will be presented.…”

Section: Applications Of Lc×lc To Phenolic Analysismentioning

confidence: 99%