Development of an integrated approach for evaluation of 2‐D gel image analysis: Impact of multiple proteins in single spots on comparative proteomics in conventional 2‐D gel/MALDI workflow

Abstract

With 2-D gel mapping, it is often observed that essentially identical proteins migrate to different positions in the gel, while some seemingly well-resolved protein spots consist of multiple proteins. These observations can undermine the validity of gel-based comparative proteomic studies. Through a comparison of protein identifications using direct MALDI-TOF/ TOF and LC-ESI-MS/MS analyses of 2-D gel separated proteins from cauliflower florets, we have developed an integrated approach to improve the accuracy and reliability of comparative 2-D electrophoresis. From 46 spots of interest, we identified 51 proteins by MALDI-TOF/TOF analysis and 108 proteins by LC-ESI-MS/MS. The results indicate that 75% of the analyzed spots contained multiple proteins. A comparison of hit rank for protein identifications showed that 37 out of 43 spots identified by MALDI matched the top-ranked hit from the ESI-MS/MS. By using the exponentially modified protein abundance index (emPAI) to determine the abundance of the individual component proteins for the spots containing multiple proteins, we found that the top-hit proteins from 40 out of 43 spots identified by MALDI matched the most abundant proteins determined by LC-MS/MS. Furthermore, our 2-D-GeLC-MS/MS results show that the top-hit proteins in 44 identified spots contributed on average 81% of the spots' staining intensity. This is the first quantitative measurement of the average rate of false assignment for direct MALDI analysis of 2-D gel spots using a new integrated workflow (2-D gel imaging, "2-D GeLC-MS/MS", and emPAI analysis). Here, the new approach is proposed as an alternative to traditional gel-based quantitative proteomics studies.

Targeting Therapy for Breast Carcinoma by ATP Synthase Inhibitor Aurovertin B

“…[34][35][36][37] As a result, the screening for disease markers became one of the main topics in a large number of proteomic studies in the past decade. [38][39][40][41][42] In this study, we performed a proteomic analysis to investigate tumor-specific protein expression of human breast carcinoma tissues.…”

Aryl Hydrocarbon Receptor Activation by Dioxin Targets Phosphoenolpyruvate Carboxykinase (PEPCK) for ADP-ribosylation via 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-inducible Poly(ADP-ribose) Polymerase (TiPARP)

“…Bands spanning 63 kDa (about 4 mm) were excised and submitted for proteomic analysis (Cornell Proteomics Facility, Cornell University, Ithaca). The SDS-gel slices were subjected to in-gel digestion by trypsin and subsequent extraction (38). The digest was reconstituted in 10 l of 2% acetonitrile with 0.5% formic acid for nano-LC-ESI-MS/MS analysis using a UltiMate3000 nano-LC system coupled to a LTQ-Orbitrap Velos mass spectrometer (Thermo-Fisher Scientific, San Jose, CA) equipped with a "CorConneX" nano ion source device (CorSolutions LLC, Ithaca, NY).…”

“…Only scores for the peptides defined by Mascot probability analysis significantly greater than "identity" were considered for peptide identification. The exponentially modified protein abundance index (emPAI) number outputted directly from Mascot results for each identified proteins was used for estimation of relative protein amounts within the same gel bands (38).…”

Reduction of Oxalate Levels in Tomato Fruit and Consequent Metabolic Remodeling Following Overexpression of a Fungal Oxalate Decarboxylase

“…In an attempt to minimize comigration of multiple protein species, we also used a relatively narrow pH range (4-7) for isoelectric focusing because the use of narrow-range immobilized pH gradient strips facilitates higher resolution (McGregor and Dunn, 2006;Sghaier-Hammami et al, 2009). However, MALDI-MS/MS is known to identify the most prevalent protein present in a gel spot sample (Jun et al, 2012), and the top-ranked hit resulting from LC-MS/MS analysis has also been shown typically to correspond to the most abundant protein among multiple proteins present in a spot (Yang et al, 2007). The spot intensities of different protein constituents were determined using the protein abundance index and the exponentially modified protein abundance index (emPAI; Supplemental Table S3), which have been routinely applied in proteomics workflows (Perkins et al, 1999;Ishihama et al, 2005;Zhang et al, 2006;Yang et al, 2007;Cilia et al, 2011;Taylor et al, 2011).…”