Does filter-aided sample preparation provide sufficient method linearity for quantitative plant shotgun proteomics?

Area of Science:

• Proteomics
• Plant Biology
• Analytical Chemistry

Background:

• Gel-free LC-based shotgun proteomics is the gold standard for proteome analysis.
• Sample preparation is a critical bottleneck affecting protein quantification accuracy.
• Detergents improve sample preparation but interfere with chromatography and ionization.

Purpose of the Study:

• To evaluate the applicability and performance of filter-aided sample preparation (FASP) for quantitative plant proteomics.
• To assess the linearity of protein quantification methods in plant matrices using FASP.
• To compare FASP with different plant-specific protein isolation protocols.

Main Methods:

• Filter-aided sample preparation (FASP) was applied to pea and Arabidopsis proteomes.
• Spiking experiments with bovine serum albumin (BSA) were conducted to evaluate linearity and sensitivity.
• Two plant protein isolation protocols (phenol extraction and detergent-based) were compared with conventional FASP.

Main Results:

• FASP demonstrated high sensitivity (LODs up to 4 fmol) and a linear dynamic range (LDR) of at least 0.6 × 10^2.
• Phenol extraction showed slightly better recoveries, while the detergent-based method exhibited better linearity.
• Plant matrices had a limited impact on FASP performance in quantitative proteomics.

Conclusions:

• FASP is highly applicable for quantitative shotgun proteomics in plant samples.
• The choice of protein isolation method has a limited effect on FASP's overall performance.
• FASP provides reliable and sensitive proteome analysis for plant research.