Quality Assessments of Long-Term Quantitative Proteomic Analysis of Breast Cancer Xenograft Tissues

Abstract

Clinical proteomics requires large-scale analysis of human specimens to achieve statistical significance. We evaluated the long-term reproducibility of an iTRAQ (isobaric tags for relative and absolute quantification)-based quantitative proteomics strategy using one channel for reference across all samples in different iTRAQ sets. A total of 148 liquid chromatography tandem mass spectrometric (LC–MS/MS) analyses were completed, generating six 2D LC–MS/MS data sets for human-in-mouse breast cancer xenograft tissues representative of basal and luminal subtypes. Such large-scale studies require the implementation of robust metrics to assess the contributions of technical and biological variability in the qualitative and quantitative data. Accordingly, we derived a quantification confidence score based on the quality of each peptide-spectrum match to remove quantification outliers from each analysis. After combining confidence score filtering and statistical analysis, reproducible protein identification and quantitative results were achieved from LC–MS/MS data sets collected over a 7-month period. This study provides the first quality assessment on long-term stability and technical considerations for study design of a large-scale clinical proteomics project.

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

“…Fewer steps are needed for the protein digestion and the chaotropic agent can be easily removed before the MS analysis, making it a favorable choice for many laboratories. 16–19 However, the method is limited by the low extraction efficiency of membrane proteins, which restricts its application for biological scaffolds as well as glycoproteins. 20 …”

Quantitative proteomics using SILAC‐MS identifies N‐acetylcysteine‐solution‐triggered reversal response of renal cell carcinoma cell lines

“…2D‐LC separation was performed on a multidimensional liquid chromatography system (Eksigent, currently part ABSCIEX, Dublin, CA) as described previously . The MS was carried out on LTQ‐Orbitrap XL (Thermo Fisher) and buffer solutions and salt elution gradients were used, as described in Zhou et al…”

“…2.8 | 2D-LC-MS/MS analysis 2D-LC separation was performed on a multidimensional liquid chromatography system (Eksigent, currently part ABSCIEX, Dublin, CA) as described previously. 16 The MS was carried out on LTQ-Orbitrap XL (Thermo Fisher) and buffer solutions and salt elution gradients were used, as described in Zhou et al 17 2.9 | Bioinformatics analysis and interpretation Thermo Fisher Scientific Proteome Discoverer software (v1.4.1) was used to create normalized ratios of heavy/light amino acids. When comparing cells in the NAC solution against the control cells, an average normalized heavy/light ratios of three biological replicates (average fold-changes) was calculated and the overexpressed proteins (average fold-changes > 1) and underexpressed proteins (average fold-changes < 1) were identified in the two cell lines, respectively.…”

Orthogonal proteomic platforms and their implications for the stable classification of high-grade serous ovarian cancer subtypes

“…These studies employed data-dependent acquisition (DDA) mass spectrometry, a mode of MS/MS data collection wherein a fixed number of precursor ions whose m/z values were recorded in a survey scan are selected for fragmentation using a pre-determined set of rules (Mann et al, 2001). DDA-based proteomic workflows have undergone considerable optimization to improve the reliability and reproducibility of the generated data in an effort to minimize the limitations due to the stochastic nature of precursor ion selection and low sampling efficiency resulting in missing values across data sets (Mertins et al, 2018; Revesz et al, 2018; Tabb et al, 2016; Zhou et al, 2017).…”