Liquid chromatography-multiple reaction monitoring mass spectrometry of peptides using stable isotope dilution (SID) provides a powerful tool for targeted protein quantitation. However, the high cost of labeled peptide standards for SID poses an obstacle to multiple reaction monitoring studies. We compared SID to a labeled reference peptide (LRP) method, which uses a single labeled peptide as a reference standard for all measured peptides, and a label-free (LF) approach, in which quantitation is based on analysis of un-normalized peak areas for detected MRM transitions. We analyzed peptides from the Escherichia coli proteins alkaline phosphatase and ␤-galactosidase spiked into lysates from human colon adenocarcinoma RKO cells. We also analyzed liquid chromatography-multiple reaction monitoring mass spectrometry data from a recently published interlaboratory study by the National A rapidly evolving approach to protein quantitation is the targeted analysis of representative peptides by liquid chromatography-tandem mass spectrometry by multiple reaction monitoring (LC-MRM-MS) 1 analysis (1-3). In this approach, peptides are quantified by monitoring several MRM transitions for each peptide with either a triple quadrupole or a quadrupole-ion trap instrument. Stable isotope dilution (SID), in which labeled peptides are used as internal standards is considered the gold standard for rigorous quantitation by LC-MRM-MS (1, 4, 5). In contrast to antibody-based quantitation, where antibody availability and specificity are often limiting, LC-MRM-MS enables configuration of an assay for essentially any protein. In practice, this approach has proven sensitive enough to apply to challenging protein quantitation problems. For example, proteins can be quantified at singledigit copy numbers in cells (6) and in plasma at levels approaching ng/ml (7,8). With antibody-based enrichment, LC-MRM-MS can achieve even greater sensitivity (9 -12).

We found a serum proteomic profile that discriminates lung cancer from matched controls. Proteomic analysis of unfractionated serum may have a role in the noninvasive diagnosis of lung cancer and will require methodological refinements and prospective validation to achieve clinical utility.

Purpose Chromosome 3q26-29 is a critical region of genomic amplification in lung squamous cell carcinomas (SCCs). Identification of candidate drivers in this region could help uncover new mechanisms in the pathogenesis and potentially new targets in SCC of the lung. Experimental Design We performed a meta-analysis of seven independent data sets containing a total of 593 human primary SCC tumor samples to identify consensus candidate drivers in 3q26-29 amplicon. Through integrating protein-protein interaction network information, we further filtered for candidates that may function together in a network. Computationally predicted candidates were validated using RNAi knock down and cell viability assays. Clinical relevance of the experimentally supported drivers was evaluated in an independent cohort of 52 lung SCC tumors using survival analysis. Results The meta-analysis identified 20 consensus candidates, among which four (SENP2, DCUN1D1, DVL3 and UBXN7) were involved in a small protein-protein interaction network. Knocking down any of the four proteins led to cell growth inhibition of the 3q26-29 amplified SCC. Moreover, knocking down of SENP2 resulted in the most significant cell growth inhibition and downregulation of DCUN1D1 and DVL3. Importantly, a gene expression signature composed of SENP2, DCUN1D1 and DVL3 stratified patients into subgroups with different response to adjuvant chemotherapy. Conclusion Together, our findings show that SENP2, DCUN1D1 and DVL3 are candidate driver genes in the 3q26-29 amplicon of SCC, providing novel insights into the molecular mechanisms of disease progression and may have significant implication in the management of SCC of the lung.

Adenocarcinomas of the lung commonly show an increase in the activity of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, yet many are resistant to apoptosis induced by the inhibition of PI3K. We hypothesized that Bcl-xL would have a synergistic effect on the apoptotic response induced by inhibition of the PI3K/Akt pathway in lung adenocarcinoma. To test this, we examined the effect of the PI3K inhibitor (LY294002) on lung adenocarcinoma cell lines expressing varying levels of Bcl-xL.