Standard Operating Procedures and Protocols for the Preparation and Analysis of Plasma Samples Using the iTRAQ Methodology

Reduced expression of mitochondrial electron transport chain proteins from hibernating hearts relative to ischemic preconditioned hearts in the second window of protection

“…However, in tandem MS they produced strong, diagnostic, low mass signature ions (m/z 113, 114, 115, 116,117, 118, 119, and 121) in each of the eight different samples including one SHAM control, which allows for relative abundance quantification from all eight samples. We compared the protein concentrations from mitochondria of the LAD region from SWOP and HIB pigs to a normal control in two 8-plex studies using the same normal control in both studies [15]. To label mitochondrial proteins from individual samples, isolates were centrifuged and 40 μg of protein has demonstrated protection during a sustained period of reduced regional blood flow.…”

Mass spectrometry based biomarker discovery, verification, and validation — Quality assurance and control of protein biomarker assays

“…The sources of variation and importance of controlling them has been demonstrated in several interlaboratory HUPO and NCI‐initiated studies (Adkins et al., 2005; Boja et al., 2011; Boja and Rodriguez, 2012; HUPO, 2010; Omenn, 2004a, b; 2007; Omenn et al., 2005; Paulovich et al., 2010; Rai et al., 2005; Rodriguez et al., 2010a, 2010b; Rudnick et al., 2010), and these sources of variability (for plasma in particular) have recently been reviewed (Gelfand and Omenn, 2011; Lista et al., 2013; Lundblad, 2005; Percy et al., 2013f; Rai and Vitzthum, 2006; Yi et al., 2011; Zhao et al., 2012). To address the issue of analytical variability, we and others have begun the development of SOPs for sample preparation and for MRM analysis (Ohlund et al., 2011; Percy et al., 2013b, 2013e; Tuck et al., 2009), and we have recently developed several “kits” to help ensure proper instrument performance (Percy et al., 2013b, 2013e). One of these kits is designed to test the instrument parameters; the other is designed to evaluate the entire workflow, from sample denaturation and digestion through the MRM data acquisition.…”

A universal molecular control for DNA, mRNA and protein expression

“…The synthetic control genes described herein represent the first set of reference standards that are both transcribed and translated, and thereby provide matched references for both mRNA and protein measurements. Unlike existing controls such as iTRAQ labelled or SIS peptides, proco peptides permit the rapid normalisation of protein extracted from any organism (as they are not designed based on organismal homology), without the use of radiolabelled peptides 20,21 . Moreover, as the proco peptides were selected based on their previous detection by LC-MS/MS, they are not impacted by insolubility 22 .…”