Comparison of Different Sample Preparation Protocols Reveals Lysis Buffer-Specific Extraction Biases in Gram-Negative Bacteria and Human Cells

Abstract

We evaluated different in-solution and FASP-based sample preparation strategies for absolute protein quantification. Label-free quantification (LFQ) was employed to compare different sample preparation strategies in the bacterium Pseudomonas aeruginosa and human embryonic kidney cells (HEK), and organismal-specific differences in general performance and enrichment of specific protein classes were noted. The original FASP protocol globally enriched for most proteins in the bacterial sample, whereas the sodium deoxycholate in-solution strategy was more efficient with HEK cells. Although detergents were found to be highly suited for global proteome analysis, higher intensities were obtained for high-abundant nucleic acid-associated protein complexes, like the ribosome and histone proteins, using guanidine hydrochloride. Importantly, we show for the first time that the observable total proteome mass of a sample strongly depends on the sample preparation protocol, with some protocols resulting in a significant underestimation of protein mass due to incomplete protein extraction of biased protein groups. Furthermore, we demonstrate that some of the observed abundance biases can be overcome by incorporating a nuclease treatment step or, alternatively, a correction factor for complementary sample preparation approaches.

Evaluation and Improvement of Quantification Accuracy in Isobaric Mass Tag-Based Protein Quantification Experiments

“…Here we used our MS1-based LFQ workflow as a reference method, as this had previously been demonstrated to reliably detect protein concentration differences across complex mixtures 22,23 In this investigation a second set of 2-proteome samples was prepared to produce two B. henselae proteome reference ratios; 1.5:1 and 2:1 and a 1:1 human proteome background (2-Proteome-Precision samples; see Experimental Section for details). All samples were acquired in triplicates using our LFQ and 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 TMT-6-plex.…”

Surfactant and Chaotropic Agent Assisted Sequential Extraction/On-Pellet Digestion (SCAD) for Enhanced Proteomics

“…Glatter et al compared the global proteome extraction differences between in-solution and FASP-based sample preparation strategies. 22 Both bacteria and human cells were investigated in the study where the inconsistency of extraction efficiency was observed between two sample types. Although the results provide more insights into the organism specific quantitative bias from different sample preparation methods, the LC-MS method was not optimized for maximizing the peptide identification (ID) and quantification, rendering less than half of the proteins that were identified from the entire proteome.…”

An effective placental cotyledons proteins extraction method for 2D gel electrophoresis

“…There is no universal protein extraction method which enables the successful extraction of the total protein from all types of human tissues . The RHE method and c T/AHE method were originally selected to study the efficiency of these methods to extract proteins from placental cotyledons tissues.…”

“…C, red box, blue box, green box, and yellow box). This might be due to selected protein lysis buffer which is coupled with selected protein precipitation methods required to extract certain proteins .…”