Comparison of fractionation strategies for offline two-dimensional liquid chromatography tandem mass spectrometry analysis of proteins from mouse adipose tissue

Abstract

a b s t r a c tIn the frame of protein identification from mouse adipose tissue, two strategies were compared for the offline elution of peptides from a strong cation exchange (SCX) column in two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) analyses. First, the salt gradient (using K + as displacing agent) was evaluated from 25 to 500 mM KCl. Then, a less investigated elution mode using a pH gradient (using citric acid and ammonium hydroxide) was carried out from pH 2.5 to 9.0. Equal amounts of peptide digest derived from mouse adipose tissue were loaded onto the SCX column and fractionated according to the two approaches. A total of 15 fractions were collected in two independent experiments for each SCX elution strategy. Then, each fraction was analyzed on a nanoLC-MS/MS platform equipped with a column-switching unit for desalting and enrichment. No substantial differences in peptide quality characteristics (molecular weight, isoelectric point, or GRAVY [grand average of hydropathicity] index distributions) were observed between the two datasets. The pH gradient approach was found to be superior, with 27.5% more unique peptide identifications and 10% more distinct protein identifications compared with the salt-based elution method. In conclusion, our data imply that the pH gradient SCX fractionation is more desirable for proteomics analysis of entire adipose tissue.Ó 2015 Elsevier Inc. All rights reserved.White adipose tissue (WAT) 3 is a complex metabolic and endocrine organ that secrets a variety of hormones, termed adipokines, into the circulation and thus regulates glucose, lipid, and energy homeostasis in mammalian organisms [1]. Pathological alterations in these various functions are well-established risk factors for metabolic disorders such as obesity, insulin resistance, and type 2 diabetes [2,3]. Investigations aimed at uncovering modifications in WAT protein expression are of important clinical interest in the quest to identify new therapeutic targets or candidate biomarkers for these prevalent pathologies [4,5]. Scientific prominence of adipose tissue proteomics along with an increasing number of adipose tissue proteomics studies per year [6][7][8][9][10][11][12][13][14] was recently observed by Peinado and coworkers [15]. The main hurdles in the isolation and identification of adipose tissue proteins are its high lipid content combined with low protein amount and a high proteome complexity related to significant tissue vascularization and the presence of a multitude of functionally important cell types located in different tissue fractions [4,16,17].Reducing protein sample complexity in two-dimensional liquid chromatography (2D-LC) investigations is a crucial issue. To this effect, various techniques of peptide separation prior to reverse phase liquid chromatography (RPLC) hyphenated to mass spectrometry (MS) detection are employed: electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) [18,19], hydrophilic interaction chromatography (HILIC) [20], ion exch...

Evaluation of Peptide Fractionation and Native Digestion as Two Novel Sample Preparation Workflows to Improve HCP Characterization by LC–MS/MS

“…Both the proteomics field as well as the HCP field made a lot of effort to evolve mass spectrometry methodologies to gain more information about the sample of interest. ,, Targeted MS provides a highly sensitive strategy to accomplish the extremely wide dynamic range and track proteins at a very low level. However, this technique requires prior knowledge of the sample content and for HCP analytics a broadly applicable approach to detect unknown proteins such as DDA/DIA is in most cases preferred. , Higher sensitivity for DDA has been achieved by offline and online multidimensional chromatography, concatenation pooling, and HCP enrichment by affinity chromatography. − Despite the uncontested success of these strategies, we provide two very powerful tools to facilitate a simpler and more efficient sample preparation for HCP identification with the same advantages. The PreOmics approach itself has a fast, reproducible, robust, and well-established protocol for proteomic applications. ,, In our studies we showed the great value of iST based methods for HCP profiling by increased sensitivity and number of proteins identified.…”

“…One widely practiced approach is the introduction of multidimensional chromatography. Orthogonal separation of tryptic peptides increases the dynamic range and reduces sample complexity enabling more sensitive detection. , Both offline and online strategies have been established as well as coupling different separation methods such as strong cation exchange combined with RP or preferably the combination of RP/RP at high/low pH. − Even further enhancement with regard to number of peptides and proteins identified was obtained by implementing a concatenation pooling step between the first and second dimension. , This concept has already been successfully applied to HCP analysis and provides a robust detection of residual HCPs down to a level of 10 ppm . Alternatively, dynamic range issues can be faced by depletion of the antibody itself and HCP enrichment traditionally achieved by affinity chromatography. , With the same idea Huang et al recently published a novel procedure, the native digestion, enabling mAb removal upon heat treatment and precipitation …”

Quantitative proteomic analysis identified differentially expressed proteins with tail/rump fat deposition in Chinese thin- and fat-tailed lambs

“…Proteins were extracted from samples after the fatty acids were isolated with chloroform and methanol (1:2) according to the previous method [36], and then treated according to the description in Fig 1B . Briefly, tissues were ground into a fine powder in liquid nitrogen, and 200 mg from each replicate samples was extracted with the lysis buffer (8 M urea, 2 M CHAPS, 4% CHAPS, 20 mM Tris-base and 30 mM DTT). The protein extract was centrifuged at 15,000 g at 4˚C for 20 min.…”

Sample Clean‐up Strategies for ESI Mass Spectrometry Applications in Bottom‐up Proteomics: Trends from 2012 to 2016

“…The sample is passed through the SPE system and the hydrophobic analyte molecules are adsorbed on the sorbent by hydrophobic interaction. Silica‐based sorbents are generally modified with groups such as C8 and C18 and SPE systems employing these functionalities have been utilized for the desalting of abundant proteins , proteome of Xenopus laevis eggs and early stage embryos , human hepatoma cell line PLC/PRF/5 lysates , mouse adipose tissue , integral membrane proteins , and elucidation of unknown peaks found in chromatograms for dirithromycin analysis .…”