Implications of partial tryptic digestion in organic–aqueous solvent systems for bottom-up proteome analysis

Mehrnejad

Biotech and App Biochem

et al. 2015

The effect of glycerol and sorbitol on the stability of porcine pancreas trypsin was investigated in this work. Molecular dynamics simulation and thermostability results showed that trypsin has two flexible regions, and polyols (sorbitol and glycerol) stabilize the enzyme by decreasing the flexibility of these regions. Radial distribution function results exhibited that sorbitol and glycerol were excluded from the first water layer of the enzyme, therefore decrease the flexibility of the regions by preferential exclusion. Also, results showed that the stabilization effect of sorbitol is more than glycerol. This observation could be because of the larger decrease in the fluctuations of trypsin in the presence of sorbitol. We also examined the role of solvent's hydrophobicity in enzyme stabilization by sorbitol and glycerol. To do so, the thermostability of trypsin was evaluated in the presence of solvents with different hydrophobicity (methanol, ethanol, isopropanol and n-propanol) in addition to the polyols. Our results depicted that glycerol is a better stabilizer than sorbitol in the presence of hydrophobic solvents (n-propanol), whereas sorbitol is a better stabilizer than glycerol in the presence of hydrophilic solvents (methanol).

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Effect of sorbitol and glycerol on the stability of trypsin and difference between their stabilization effects in the various solvents

Mehrnejad

Biotech and App Biochem

et al. 2015

“…Organic solvent-assisted digestion was used in order to improve efficiency of trypsin digestion as previously was shown [27][28][29][30][31][32]. The peptides were eluted from the column by centrifugation at RT, 1,000 x g for 5 min.…”

Section: Methodsmentioning

confidence: 99%

Optimization of calmodulin-affinity chromatography for brain and organelles

Kulej

Palmisano

Edwards

et al. 2015

EuPA Open Proteomics

Graphical abstract fx1Highlights  Optimization of affinity enrichment of Calmodulin-binding proteins using CaM resin.  Identification of 3,529 putative CaM-BPs from mouse brain tissue.  Identification of 2,698 putative CaM-BPs from isolated nerve-terminals.  Characterization of and 2,783 unique phosphopeptides derived from 984 potential synaptosomal CaM-BPs 2 AbstractCalmodulin (CaM) is a Ca 2+ -binding signaling protein that binds to and activates many target proteins, known as calmodulin-binding proteins (CaM-BPs). They are involved in multiple cellular processes. Despite the diversity and importance of CaM-BPs, many remain to be identified and characterized. We performed extensive optimization of a CaM-affinity capture method, using commercial CaM-chromatographic material. We identify both the Ca 2+ -dependent and -independent CaM binding proteomes in both mouse brain and in rat brain neuronal organelles, synaptosomes, and compared cytosolic with membrane associated targets. Fractionation of peptides, derived from onresin tryptic digestion, using hydrophilic interaction liquid chromatography (HILIC) was combined with reversed-phase liquid chromatography tandem mass spectrometry (LC-MS/MS) to improve identification of low abundance CaM-BPs in a reproducible and sensitive manner. Various detergents were tested for the most efficient membrane protein solubilization for pull-down of membraneassociated CaM-BPs. We identified 3,529 putative mouse brain CaM-BPs, of which 1,629 were integral membrane or membrane-associated. Among them, 170 proteins were known CaM-BPs or previously reported as potential CaM-BPs while 696 contained predicted CaM binding motifs. In synaptosomes we identified 2,698 CaM-BPs and 2,783 unique phosphopeptides derived from 984 of the potential synaptosomal CaM-BPs. Overall, our improved workflow provides unmatched sensitivity for the identification of the CaM binding proteome and its associated phosphoproteome and this now enables sensitive analysis of organelle-specific CaM-BPs. 3

Mass Spectrometry Reviews

“…Large extent of organic solvents concentration suggests that the results of various buffers might be sample‐specific. For example, Hervey et al reported that 6 M GdnHCl and 80% ACN yielded a higher number of peptides than 0.1% RapiGest (Hervey, Strader, & Hurst, ), and in another study on a yeast lysate the opposite trend was was attributed to a decrease of trypsin activity (Wall et al, ).…”

Section: Biochemical Protocols and Their Impact On The Proteome Data mentioning

confidence: 99%

Proteome analysis of tissues by mass spectrometry

Đapić

Baljeu‐Neuman

Uwugiaren

et al. 2019

Tissues and biofluids are important sources of information used for the detection of diseases and decisions on patient therapies. There are several accepted methods for preservation of tissues, among which the most popular are fresh‐frozen and formalin‐fixed paraffin embedded methods. Depending on the preservation method and the amount of sample available, various specific protocols are available for tissue processing for subsequent proteomic analysis. Protocols are tailored to answer various biological questions, and as such vary in lysis and digestion conditions, as well as duration. The existence of diverse tissue‐sample protocols has led to confusion in how to choose the best protocol for a given tissue and made it difficult to compare results across sample types. Here, we summarize procedures used for tissue processing for subsequent bottom‐up proteomic analysis. Furthermore, we compare protocols for their variations in the composition of lysis buffers, digestion procedures, and purification steps. For example, reports have shown that lysis buffer composition plays an important role in the profile of extracted proteins: the most common are tris(hydroxymethyl)aminomethane, radioimmunoprecipitation assay, and ammonium bicarbonate buffers. Although, trypsin is the most commonly used enzyme for proteolysis, in some protocols it is supplemented with Lys‐C and/or chymotrypsin, which will often lead to an increase in proteome coverage. Data show that the selection of the lysis procedure might need to be tissue‐specific to produce distinct protocols for individual tissue types. Finally, selection of the procedures is also influenced by the amount of sample available, which range from biopsies or the size of a few dozen of mm2 obtained with laser capture microdissection to much larger amounts that weight several milligrams.