When the pH is greater than the isoelectric point of the protein, shouldn't the protein carry a negative charge? Why is it not detectable in negative mode using mass spectrometry?

Yes, when the pH is greater than the isoelectric point (pI) of a protein, the protein will carry a negative charge. This is because, under these conditions, the positively charged amino acid residues (such as lysine, arginine, and histidine) lose their protons, while the negatively charged amino acid residues (such as aspartic acid and glutamic acid) retain their negative charge. As a result, the entire protein carries a negative charge.

However, in mass spectrometry analysis, the negative mode is rarely used for protein detection for the following reasons:

1. Electrospray Ionization (ESI):

In protein mass spectrometry, the most commonly used ionization method is electrospray ionization (ESI). The effect of ESI in the positive mode is generally better than in the negative mode because it is easier to produce multiply protonated protein ions in the positive mode. These multiply protonated ions have a lower mass-to-charge ratio, making it easier for the mass analyzer to detect.

2. Fragmentation Efficiency:

For the negative mode, fragmentation efficiency is usually lower. This means it is more challenging to generate sufficient information to identify peptide fragments of the protein sequence in the negative mode. In contrast, the positive mode has higher fragmentation efficiency, producing more useful information.

3. Compatibility of Ion Source and Mass Analyzer:

Many mass spectrometry instruments perform better in the positive mode. The negative mode may require changing instrument parameters, such as ion source voltage and gas flow rate, which may lead to reduced sensitivity or signal distortion.

Therefore, even though proteins carry a negative charge at pH conditions above their isoelectric point, the positive mode is typically used for protein detection in mass spectrometry because it offers higher sensitivity and fragmentation efficiency.

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