De Novo Sequencing Sample Quantity

De novo sequencing, i.e., sequencing from scratch, is a method for the sequence analysis of proteins or peptides, particularly useful when the reference sequence is absent or incomplete. This technique primarily relies on mass spectrometry (MS) data, inferring the original sequence of a protein or peptide by analyzing the mass spectra of peptide ions. De Novo sequencing is critically important in fields such as studying novel proteins, protein engineering, drug discovery, and identification of biomarkers. However, conducting high-quality De Novo sequencing requires meeting certain sample quantity requirements, a key factor in ensuring the accuracy and validity of the sequencing data.

 

Sample Submission Requirements

1. Sample size requirements

(1) Minimum sample quantity

The minimum sample quantity for De Novo sequencing usually depends on the sensitivity of the mass spectrometer, the purity of the sample, and the complexity of the sequencing target. For modern high-sensitivity mass spectrometry equipment, a few picomoles (pmol) of peptides or proteins may be sufficient for analysis. However, in practical applications, the recommended minimum sample quantity usually ranges from a few hundred picomoles to a few nanomoles (nmol), to compensate for losses during sample preparation and ensure adequate signal strength.

(2) Ideal sample quantity

To obtain high-quality mass spectrometry data and improve sequence coverage, the ideal sample quantity should be higher. Usually, 1-10 nanomoles of peptides or proteins are the ideal starting quantity for De Novo sequencing. Such quantity allows for repeated experiments, ensuring the repeatability and accuracy of the results.

(3)  Sample purity

The purity of the sample is also a crucial factor for a successful experiment. High-purity samples can reduce background noise and improve the detection sensitivity of specific peptide signals. Therefore, when determining the required sample quantity, the purification and preprocessing steps of the sample also need to be considered.

 

2. Factors That Affect Sample Quantity Requirements

(1) Sensitivity of mass spectrometer

Higher-sensitivity mass spectrometry equipment can analyze smaller quantities of samples, but also requires higher sample purity.

(2) Complexity of sequencing target

The higher the complexity of the target protein, the greater the sample quantity required. For proteins with multiple glycosylation sites or other post-translational modifications, more samples may be needed to ensure adequate coverage and resolution.

(3) Availability and cost of the sample

In some cases, the availability and cost of the sample may also affect the actual available sample quantity. For samples that are hard to acquire or costly, researchers may need to use as little sample quantity as possible for the experiment.