Proteome Coverage after Simultaneous Proteo-Metabolome Liquid-Liquid Extraction

Abstract

Proteomics and metabolomics are essential in systems biology, and simultaneous proteo-metabolome liquid-liquid extraction (SPM-LLE) allows isolation of the metabolome and proteome from the same sample. Since the proteome is present as a pellet in SPM-LLE, it must be solubilized for quantitative proteomics. Solubilization and proteome extraction are critical factors in the information obtained at the proteome level. In this study, we investigated the performance of two surfactants (sodium deoxycholate (SDC), sodium dodecyl sulfate (SDS)) and urea in terms of proteome coverage and extraction efficiency of an interphase proteome pellet generated by methanol-chloroform based SPM-LLE. We also investigated how the performance differs when the proteome is extracted from the interphase pellet or by direct cell lysis. We quantified 12 lipids covering triglycerides and various phospholipid classes, and 25 polar metabolites covering central energy metabolism in chloroform and methanol extracts. Our study reveals that the proteome coverages between the two surfactants and urea for the SPM-LLE interphase pellet were similar, but the extraction efficiencies differed significantly. While SDS led to enrichment of basic proteins, which were mainly ribosomal and ribonuclear proteins, urea was the most efficient extraction agent for simultaneous proteo-metabolome analysis. The results of our study also show that the performance of surfactants for quantitative proteomics is better when the proteome is extracted through direct cell lysis rather than an interphase pellet. In contrast, the performance of urea for quantitative proteomics was significantly better when the proteome was extracted from an interphase pellet than by direct cell lysis. We demonstrated that urea is superior to surfactants for proteome extraction from SPM-LLE interphase pellets, with a particularly good performance for the extraction of proteins associated with metabolic pathways. Data are available via ProteomeXchange with identifier PXD027338.

Keywords: SP3; bottom-up proteomics; in-solution digest; label free quantification; mass spectrometry; metabolomics; proteomics; sample preparation; simultaneous proteo-metabolomics.

Figure 1

Quantification of selected lipids and polar metabolites extracted from the chloroform phase and methanol phase of a simultaneous proteo-metabolomic liquid–liquid extraction. (A, B) Violin plots showing the coefficient of variation [%] for all lipid analytes (A) and sorted based on lipid class and for the internal standards (ISTD, DMPC: 1,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPE: 1,2-dimyristoyl-sn-glycero-3-phosphorylethanolamine, TMG: 1,2,3-trimyristoyl-glycerol) (B). (C) Color-coded representation of the coefficient of variation [%] of the different lipid analytes. LPE: lysophosphatidylethanolamine, PC: phosphatidylcholine, PI: phosphatidylinositol, TAG: triglyceride. (D) Violin plots showing the coefficient of variation [%] for internal standards (ISTD), extracellular and intracellular polar metabolites. (E) Color-coded representation of the coefficient of variation [%] of the ¹³C labeled yeast metabolites used as internal standards (ISTD), extracellular and intracellular polar metabolites. n = 5 independent experiments (biological replicates).

Figure 2

Protein yield (A), number of identified proteins (B, C), and relative percentage of missed cleavages after tryptic digestion (D) detected in the proteome solubilized from the interphase pellets. (A) Protein yield (μg) and (B) number of identified proteins from the interphase pellets solubilized by sodium deoxycholate (SDC, green), sodium dodecyl sulfate (SDS, orange), and urea (purple). (C) Number of proteins reproducibly identified in all replicates (n = 5) with the indicated buffer systems. (D) Relative percentage of missed cleavages after tryptic digestion of the interphase pellet extracted by SDC (green), SDS (orange), and urea (purple) based buffer systems. (A, B, D) Bar graph: mean with standard deviation, statistical analysis: two-tailed unpaired t-test. n = 5 independent experiments (biological replicates). Average number of cells: 4.3 × 10⁶ cells.

Figure 3

Quantitative analysis of proteins extracted from interphase pellets of simultaneous proteo-metabolomics liquid–liquid extractions (SPM-LLE). (A) Principal component analysis of proteins extracted from the SPM-LLE interphase pellets using sodium deoxycholate (SDC, green), sodium dodecyl sulfate (SDS, orange), or urea (purple). Protein abundance levels, reproducibly quantified in all independent experiments and conditions, were used as input for PCA. (B) Comparison of efficiency to extract proteomes from the SPM-LLE interphase pellet with SDC (green), SDS (orange), or urea (purple). Significance threshold for enrichment: adjusted p-value ≤0.05 (two-tailed unpaired t-test, Benjamini–Hochberg correction), fold change (FC) of 1.5: colored transparent dots, FC of ≥2: colored dots. (C) Physicochemical properties of proteins enriched in the SPM-LLE interphase pellets (colored solid lines, FC ≥ 1.5, adjusted p-value ≤0.05) as compared to the human reference proteome (SwissProt, uniprot.org, black dashed line). n = 5 independent experiments (biological replicates).

Figure 4

Protein yield (A), number of proteins identified (B, C), and relative percentage of missed cleavages after tryptic digestions (D) obtained by direct cell lysis. (A) Protein yield (μg) and (B) number of identified proteins by direct cell lysis using sodium deoxycholate (SDC), sodium dodecyl sulfate (SDS), and urea. (C) Number of proteins reproducibly identified in all biological replicates (n = 5) with the indicated extraction agents. (D) Relative percentage of missed cleavages after tryptic digestion of the proteome extracted by direct cell lysis using SDC (green), SDS (orange) and urea (purple) based buffer systems. (A, B, D) Bar graph: mean with standard deviation. Statistical analyses with two-tailed unpaired t-test. n = 5 independent experiments (biological replicates). Average number of cells: 4.3 × 10⁶ cells.

Figure 5

Comparison of proteins extracted from simultaneous proteo-metabolomics liquid–liquid extraction (SPM-LLE) interphase pellets versus direct cell lysis. (A) Number of identified proteins in the proteomes extracted from interphases of SPM-LLE (same data as shown in Figure 2B) and by direct cell lysis (same data as shown in Figure 4B) using SDC, SDS and urea. Mean with standard deviation. (B) Venn diagrams showing the number of proteins reproducibly identified in all replicates using SDC, SDS, and urea. Statistical analyses were performed using the two-tailed unpaired t test. n = 5 independent experiments (biological replicates). (C) Principal component analysis of proteins extracted from SPM-LLE interphase pellet (triangles) or direct cell lysis (circles). Protein abundance levels, reproducibly quantified in all independent experiments and conditions, were used as input for PCA. (D) Comparison of efficiency to extract proteomes from the SPM-LLE interphase pellets versus direct cell lysis. Significance threshold for enrichment: adjusted p-value ≤0.05 (two-tailed unpaired t-test, Benjamini–Hochberg correction), fold change (FC) of 1.5: colored transparent dots, FC of ≥2: colored dots. (E) Physicochemical properties of enriched proteins (FC ≥ 1.5, p-value ≤0.05). Proteins from SPM-LLE interphase pellets are represented with colored dashed lines, proteins from direct cell lysis are represented as colored solid lines. Physicochemical properties of the human reference proteome (SwissProt, uniprot.org) are shown with a black dashed line. Sodium deoxycholate (SDC, green), sodium dodecyl sulfate (SDS, orange), or urea (purple) containing buffer. n = 5 independent experiments (biological replicates).