Why Are Peptides Lost After C18 SPE Desalting of Casein Hydrolysate Before LC-MS
1. Sample Processing
Potential complications during sample preparation may contribute to peptide loss. For instance, incomplete binding of peptides to the C18 column or inefficient elution may occur. These issues can arise when sample conditions—such as pH, ionic strength, or organic solvent content—are suboptimal for effective peptide retention and release on the C18 sorbent.
2. Selection of C18 Column
C18 columns vary in properties such as particle size, pore diameter, and surface chemistry. These physicochemical characteristics directly impact peptide binding and elution efficiency. A mismatch between the column specifications and sample characteristics may result in poor peptide recovery.
3. Selection of Eluent
The elution solvent plays a critical role in peptide recovery. Typically, eluents containing a defined proportion of organic solvents (e.g., acetonitrile or methanol) enhance peptide elution. However, excessive organic content may compromise peptide solubility, thereby reducing overall recovery.
4. Properties of Peptides
Peptide-specific properties—including molecular size, net charge, and hydrophobicity—also influence their interaction with the C18 matrix. In some cases, the peptides generated from casein hydrolysis may not exhibit suitable characteristics for retention and elution using a conventional C18 SPE approach.
5. Operational Issues
Technical factors during experimental handling may also contribute to peptide loss. For example, rapid elution flow rates can lead to incomplete peptide elution. Additionally, mechanical losses such as sample evaporation or accidental splashing during transfer may further reduce recovery.
To improve peptide recovery, the following strategies may be considered:
1. Optimize the composition of the elution buffer by increasing the concentration of the organic solvent.
2. Employ freshly activated or high-performance SPE columns.
3. Adjust the sample pH and salt concentration based on the physicochemical properties of the peptides.
4. Explore alternative SPE sorbents, such as more polar columns, to improve peptide retention and elution efficiency.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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