Which of the following techniques can be used to identify a post-translational

modification such as phosphorylation or methylation of a protein, and the

residue where it is modified?

2-D gel electrophoresis

Size exclusion chromatography

Mass spectrometry

UV-Visible spectroscopy

Mass spectrometry is the primary technique for identifying post-translational modifications (PTMs) like phosphorylation or methylation on specific protein residues, as it measures mass shifts and sequences peptides to pinpoint modifications. Among the options—2-D gel electrophoresis, size exclusion chromatography, mass spectrometry, and UV-Visible spectroscopy—only mass spectrometry provides residue-level precision.

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2-D Gel Electrophoresis for PTMs

2-D gel electrophoresis separates proteins by isoelectric point and molecular weight, revealing PTM-induced shifts in spot position due to charge or size changes from phosphorylation or glycosylation. It detects potential modifications indirectly through comparative analysis but requires follow-up mass spectrometry for residue identification, limiting standalone use. This method suits initial screening in complex samples yet lacks site-specificity.

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Size Exclusion Chromatography Explained

Size exclusion chromatography (SEC) separates proteins by hydrodynamic volume, useful for assessing PTM effects on overall protein size like heavy glycosylation. It cannot identify specific modifications or residues, as it ignores charge or mass differences from phosphorylation/methylation. SEC supports purification before advanced analysis but fails for direct PTM localization.

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Mass Spectrometry: The Gold Standard

Mass spectrometry excels by fragmenting peptides and detecting mass-to-charge ratios, identifying PTMs via signature mass shifts—e.g., +80 Da for phosphorylation or +14 Da for methylation—and localizing them to residues through tandem MS. Techniques like LC-MS/MS enable high-throughput, sensitive detection even in low-abundance proteins. It outperforms others for comprehensive PTM mapping in proteomics workflows.

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UV-Visible Spectroscopy Limitations

UV-Visible spectroscopy measures protein absorbance at 280 nm for concentration or monitors structural changes but cannot distinguish PTMs or residues. Phosphorylation or methylation rarely alters UV spectra distinctly from unmodified proteins. This technique serves quantification, not identification.

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