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. 2022 Jul 18;94(29):10320–10328. doi: 10.1021/acs.analchem.1c04200

Figure 3.

Figure 3

Independent method validations and complex applications of SP4 cleanup for proteomics. The SP4 protocol was provided to three collaborators and applied to several sample types to compare SP4 with SP3. (A) Lab 1 performed SP3 with either SpeedBeads carboxylate or ReSyn HILIC magnetic beads compared with overnight acetone (ACT(O/N)) precipitation and SP4-GB for 1, 10, and 250 μg preparations of Jurkat human immortalized T cell lysate (n = 3). †n = 2; see Supporting Methods. (B) Acetone precipitation and SP4 were compared for 250 μg HEK293 lysate digested with trypsin +/− Lys-C. (C) Lab 2 processed 25 μg of HEK293 lysate for SP3, SP4-BF, and SP4-GB protocols. (D) Lab 3 processed two independent n = 5 comparisons of SP3 and SP4-GB using 50 μg of E14 murine embryonic stem cell lysate. (E) SP3 and SP4 preparations of more complex lysates/homogenates derived from whole organs, organisms, and formalin-fixed paraffin-embedded (FFPE) tissue. Bar charts present median and standard deviation, with significance assessed by ANOVA (A, C) and t-test (B, D, E). Protein coefficients of variance distributions represented by violin plot (thick line–median, thin lines–quartiles). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns–not significant.