Evaluation of FASP, SP3, and iST Protocols for Proteomic Sample Preparation in the Low Microgram Range

Abstract

Efficient and reproducible sample preparation is a prerequisite for any robust and sensitive quantitative bottom-up proteomics workflow. Here, we performed an independent comparison between single-pot solid-phase-enhanced sample preparation (SP3), filter-aided sample preparation (FASP), and a commercial kit based on the in-StageTip (iST) method. We assessed their performance for the processing of proteomic samples in the low μg range using varying amounts of HeLa cell lysate (1-20 μg of total protein). All three workflows showed similar performances for 20 μg of starting material. When handling sample sizes below 10 μg, the number of identified proteins and peptides as well as the quantitative reproducibility and precision drastically dropped in case of FASP. In contrast, SP3 and iST provided high proteome coverage even in the low μg range. Even when digesting 1 μg of starting material, both methods still enabled the identification of over 3000 proteins and between 25 000 and 30 000 peptides. On average, the quantitative reproducibility between experimental replicates was slightly higher in case of SP3 (R = 0.97 (SP3); R = 0.93 (iST)). Applying SP3 toward the characterization of the proteome of FACS-sorted tumor-associated macrophages in the B16 tumor model enabled the quantification of 2965 proteins and revealed a "mixed" M1/M2 phenotype.

Fibroblast Growth Factor 2‐Mediated Regulation of Neuronal Exosome Release Depends on VAMP3/Cellubrevin in Hippocampal Neurons

“…Afterward, the reaction was quenched by adding another 9 µL of 200 m m dithiothreitol. Proteolytic digestion was performed using a modified protocol for single‐pot solid‐phase enhanced sample preparation (SP3) . Briefly, after binding of proteins to 40 µg of a 1:1 mixture of hydrophilic and hydrophobic magnetic Sera‐Mag SpeedBeads (GE Healthcare) with a final concentration of 70% acetonitrile for 30 min at room temperature, beads were washed twice with 200 µL 70% ethanol and twice with 180 µL acetonitrile.…”

Improving Proteome Coverage for Small Sample Amounts: An Advanced Method for Proteomics Approaches with Low Bacterial Cell Numbers

“…[17,18] Sielaff et al published an slightly improved protocol in 2017 that enabled identification of about 3400 proteins and up to 30 000 peptides from as low as 1 µg protein from HeLa cells. [19] However, so far no studies are available that apply the SP3 protocol to bacteria. When only low numbers of bacteria can be recovered it is crucial to use a harsh but efficient cell disruption, without interfering with subsequent sample preparation.…”

“…The SP3 protocol provides the option to easily remove compounds required for efficient cell disruption but incompatible with subsequent analysis by MS. In order to proof applicability to diverse bacteria, we combined the SP3 protocol introduced by Sielaff et al [19] with optimized cell disruption protocols for the Gram-negative bacterium Legionella pneumophila Corby, the non-capsulated Gram-positive pathogen Staphylococcus aureus, and the capsulated Gram-positive veterinary pathogen Streptococcus suis. In all three cases, we observed increased peptide coverage and thus, enhanced protein identification as well as more reliable and reproducible protein quantification with the adapted SP3 protocol in comparison to the strain specific standard protein preparation and in-solution digestion procedures applied before.…”

“…Sielaff et al. published an slightly improved protocol in 2017 that enabled identification of about 3400 proteins and up to 30 000 peptides from as low as 1 µg protein from HeLa cells . However, so far no studies are available that apply the SP3 protocol to bacteria.…”

“…The SP3 protocol provides the option to easily remove compounds required for efficient cell disruption but incompatible with subsequent analysis by MS. In order to proof applicability to diverse bacteria, we combined the SP3 protocol introduced by Sielaff et al . with optimized cell disruption protocols for the Gram‐negative bacterium Legionella pneumophila Corby, the non‐capsulated Gram‐positive pathogen Staphylococcus aureus , and the capsulated Gram‐positive veterinary pathogen Streptococcus suis .…”