5 research outputs found
Comparative Evaluation of Small Molecular Additives and Their Effects on Peptide/Protein Identification
Detergents
and salts are widely used in lysis buffers to enhance
protein extraction from biological samples, facilitating in-depth
proteomic analysis. However, these detergents and salt additives must
be efficiently removed from the digested samples prior to LC-MS/MS
analysis to obtain high-quality mass spectra. Although filter-aided
sample preparation (FASP), acetone precipitation (AP), followed by
in-solution digestion, and strong cation exchange-based centrifugal
proteomic reactors (CPRs) are commonly used for proteomic sample processing,
little is known about their efficiencies at removing detergents and
salt additives. In this study, we (i) developed an integrative workflow
for the quantification of small molecular additives in proteomic samples,
developing a multiple reaction monitoring (MRM)-based LC-MS approach
for the quantification of six additives (i.e., Tris, urea, CHAPS,
SDS, SDC, and Triton X-100) and (ii) systematically evaluated the
relationships between the level of additive remaining in samples following
sample processing and the number of peptides/proteins identified by
mass spectrometry. Although FASP outperformed the other two methods,
the results were complementary in terms of peptide/protein identification,
as well as the GRAVY index and amino acid distributions. This is the
first systematic and quantitative study of the effect of detergents
and salt additives on protein identification. This MRM-based approach
can be used for an unbiased evaluation of the performance of new sample
preparation methods. Data are available via ProteomeXchange under
identifier PXD005405
Enhancing Membrane Protein Identification Using a Simplified Centrifugation and Detergent-Based Membrane Extraction Approach
Membrane proteins
may act as transporters, receptors, enzymes,
and adhesion-anchors, accounting for nearly 70% of pharmaceutical
drug targets. Difficulties in efficient enrichment, extraction, and
solubilization still exist because of their relatively low abundance
and poor solubility. A simplified membrane protein extraction approach
with advantages of user-friendly sample processing procedures, good
repeatability and significant effectiveness was developed in the current
research for enhancing enrichment and identification of membrane proteins.
This approach combining centrifugation and detergent along with LC-MS/MS
successfully identified higher proportion of membrane proteins, integral
proteins and transmembrane proteins in membrane fraction (76.6%, 48.1%,
and 40.6%) than in total cell lysate (41.6%, 16.4%, and 13.5%), respectively.
Moreover, our method tended to capture membrane proteins with high
degree of hydrophobicity and number of transmembrane domains as 486
out of 2106 (23.0%) had GRAVY > 0 in membrane fraction, 488 out
of
2106 (23.1%) had TMs ≥ 2. It also provided for improved identification
of membrane proteins as more than 60.6% of the commonly identified
membrane proteins in two cell samples were better identified in membrane
fraction with higher sequence coverage. Data are available via ProteomeXchange
with identifier PXD008456
Additional file 3: of Follow-up of the manganese-exposed workers healthy cohort (MEWHC) and biobank management from 2011 to 2017 in China
Table S2. Summary of the number of samples deposited in the biobank obtained from the heavy-metal cohort (MEWHC) initiated in 2011. (PDF 89 kb
Additional file 2: of Follow-up of the manganese-exposed workers healthy cohort (MEWHC) and biobank management from 2011 to 2017 in China
Supplementary information for the biobank of the MEWHC. (PDF 398 kb
Additional file 1: of Follow-up of the manganese-exposed workers healthy cohort (MEWHC) and biobank management from 2011 to 2017 in China
Table S1. Air monitoring of different workshops and jobs in the ferro-Mn alloy production plant in 2017. (PDF 107 kb