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 noted organismal specific differences in general performance and enrichment of specific protein classes. The original FASP protocol globally enriched for most proteins on the bacterial sample whereas the Sodium deoxycholate in-solution strategy was more efficient on HEK cells. Although detergents were found 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 significant underestimation of protein mass due to incomplete protein extraction of biased protein groups

INSTRUMENT(S): LTQ Orbitrap Velos, LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human) Pseudomonas Aeruginosa Pao1

TISSUE(S): Permanent Cell Line Cell, Hek-293 Cell

SUBMITTER: Timo Glatter  

LAB HEAD: Alexander Schmidt

PROVIDER: PXD001511 | Pride | 2015-10-06

REPOSITORIES: Pride