Modified filter-aided sample preparation (FASP) method increases peptide and protein identifications for shotgun...

Area of Science:

• Proteomics
• Biochemistry
• Analytical Chemistry

Background:

• Mass spectrometry (MS)-based protein identification relies heavily on effective protein extraction and digestion.
• Sodium dodecyl sulfate (SDS), a common surfactant, can hinder enzymatic digestion and MS analysis.
• Filter-aided sample preparation (FASP) was developed to remove SDS prior to MS analysis.

Purpose of the Study:

• To develop and evaluate a modified FASP (mFASP) method for improved protein identification.
• To compare the performance of mFASP with the original FASP method in shotgun proteomic experiments.

Main Methods:

• The modified FASP (mFASP) method utilizes sodium deoxycholate and a detergent removal spin column.
• The mFASP method was compared to the standard FASP method for protein and peptide identification.

Main Results:

• mFASP identified significantly more peptides (9139 ± 317) and protein groups (1377 ± 33) compared to FASP (4570 ± 392 peptides, 862 ± 46 protein groups).
• mFASP achieved higher average peptide-to-protein coverage (21.2% vs. 13.2%) and identified more hydrophobic peptides.

Conclusions:

• The mFASP method provides reliable and efficient protein and peptide identification from SDS-containing whole-cell extracts.
• This approach enhances proteomic analysis throughput, comprehensiveness, and potential for new biomarker discovery.