Why Can't Proteins Be Detected in Negative Mode Mass Spectrometry When pH Is Above Their Isoelectric Point?
When the pH exceeds the isoelectric point (pI) of a protein, the protein tends to acquire a negative charge. This occurs because positively charged amino acid residues such as lysine, arginine, and histidine lose their protons, while the negatively charged residues like aspartic acid and glutamic acid retain their charge, resulting in an overall negative charge on the protein. Despite this, negative mode is seldom employed in protein mass spectrometry for several reasons:
Electrospray Ionization (ESI)
ESI is the predominant ionization method in protein mass spectrometry. Positive mode is generally more effective than negative mode due to the ease of generating multiply protonated protein ions, which have lower mass-to-charge ratios and are easier for mass analyzers to detect.
Fragmentation Efficiency
In negative mode, fragmentation efficiency is typically lower, making it challenging to generate sufficient peptide fragment information for protein sequence identification. Conversely, positive mode yields higher fragmentation efficiency, providing more valuable data.
Instrument Compatibility
Many mass spectrometry instruments exhibit optimal performance in positive mode. Operating in negative mode may necessitate adjustments to instrument parameters, such as ion source voltage and gas flow, potentially compromising sensitivity or causing signal distortion.
Hence, despite proteins being negatively charged at a pH above their pI, positive mode is favored in mass spectrometry due to its superior sensitivity and fragmentation efficiency.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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