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Recent advances in MS instrumentation and progresses in phosphopeptide enrichment, in conjunction with more powerful data analysis tools, have facilitated unbiased characterization of thousands of site-specific phosphorylation events. Combined with stable isotope labeling by amino acids in cell culture metabolic labeling, these techniques have made it possible to quantitatively evaluate phosphorylation changes in various physiological states in stable cell lines. However, quantitative phosphoproteomics in primary cells and tissues remains a major technical challenge due to the lack of adequate techniques for accurate quantification. Here, we describe an integrated strategy allowing for large scale quantitative profiling of phosphopeptides in complex biological mixtures. In this technique, the mixture of proteolytic peptides was subjected to phosphopeptide enrichment using a titania affinity column, and the purified phosphopeptides were subsequently labeled with iTRAQ reagents. After further fractionation by strong-cation exchange, the peptides were analyzed by LC-MS/MS on an Orbitrap mass spectrometer, which collects CID and high-energy collisional dissociation (HCD) spectra sequentially for peptide identification and quantitation. We demonstrate that direct phosphopeptide enrichment of protein digests by titania affinity chromatography substantially improves the efficiency and reproducibility of phosphopeptide proteomic analysis and is compatible with downstream iTRAQ labeling. Conditions were optimized for HCD normalized collision energy to balance the overall peptide identification and quantitation using the relative abundances of iTRAQ reporter ions. Using this approach, we were able to identify 3557 distinct phosphopeptides from HeLa cell lysates, of which 2709 were also quantified from HCD scans.

Protein phosphorylation is a ubiquitous post-translational modification critical to many cellular processes. Large-scale unbiased characterization of phosphorylation status remains a major technical challenge in proteomics. In the present work, we evaluate and optimize titania-based affinity enrichment for global profiling of phosphopeptides from complex biological mixtures. We demonstrate that inclusion of glutamic acid in the sample loading buffer substantially reduced nonspecific binding of nonphosphorylated peptides to the titania while retaining the high binding affinity for phosphopeptides. The reduction in nonspecific peptide binding enhanced overall phosphopeptide recovery, ranging from 22 to 85%, and led to substantial improvement in large-scale global profiling. In addition, we observed that the overall identification of phosphopeptides was significantly enhanced by neutral loss-triggered MS (3) scans and respective use of multiple charge- and mass-dependent filtering criteria for MS (2) and MS (3) spectra. In conjunction with strong-cation exchange chromatography (SCX) for prefractionation, a total of 4002 distinct phosphopeptides were identified from SKBr3 breast cancer cells at false-positive rates of 3.7% and 5.5%, respectively, for singly and doubly phosphorylated peptides.

The pharmacokinetics of an antibody (huA1)-drug (auristatin microtubule disrupting MMAF) conjugate, targeting 5T4-expressing cells, were characterized during the discovery and development phases in female nu/nu mice and cynomolgus monkeys after a single dose and in S-D rats and cynomolgus monkeys from multidose toxicity studies. Plasma/serum samples were analyzed using an ELISA-based method for antibody and conjugate (ADC) as well as for the released payload using an LC-MS/MS method. In addition, the distribution of the Ab, ADC, and released payload (cys-mcMMAF) was determined in a number of tissues (tumor, lung, liver, kidney, and heart) in two tumor mouse models (H1975 and MDA-MB-361-DYT2 models) using similar LBA and LC-MS/MS methods. Tissue distribution studies revealed preferential tumor distribution of cys-mcMMAF and its relative specificity to the 5T4 target containing tissue (tumor). Single dose studies suggests lower CL values at the higher doses in mice, although a linear relationship was seen in cynomolgus monkeys at doses from 0.3 to 10 mg/kg with no evidence of TMDD. Evaluation of DAR (drug-antibody ratio) in cynomolgus monkeys (at 3 mg/kg) indicated that at least half of the payload was still on the ADC 1 to 2 weeks after IV dosing. After multiple doses, the huA1 and conjugate data in rats and monkeys indicate that exposure (AUC) increases with increasing dose in a linear fashion. Systemic exposure (as assessed by Cmax and AUC) of the released payload increased with increasing dose, although exposure was very low and its pharmacokinetics appeared to be formation rate limited. The incidence of ADA was generally low in rats and monkeys. We will discuss cross species comparison, relationships between the Ab, ADC, and released payload exposure after multiple dosing, and insights into the distribution of this ADC with a focus on experimental design as a way to address or bypass apparent obstacles and its integration into predictive models.

Global analysis of glycoproteins shows great promise for the discovery of therapeutic targets and clinical biomarkers. Selective capture of glycopeptides by hydrazide resin followed by mass spectrometric identification of the peptides released by PNGaseF treatment has been most widely used. However, the majority of the reports using this approach focus on global profiling, rather than relative quantitation of glycoprotein alternations in pathological states. We describe an integrated strategy allowing for relative quantitation of glycoproteins in complex biological mixtures using this approach. The strategy includes periodate oxidation of tryptic digests, solid-phase enrichment of glycopeptides via hydrazide-coupled magnetic beads, in conjunction with (18)O stable isotope labeling catalyzed by both trypsin and PNGaseF, and subsequent identification and quantitation by LC-MS/MS analysis. Three (18)O atoms ((18)O(3)) are incorporated into N-linked glycopeptides for samples treated in (18)O-water, two at the carboxyl terminus by trypsin during hydrazide coupling and the third at the N-glycosylation site through PNGaseF-mediated deglycosylation. Thus, mass shifts of 6 and 8 Da are indicative of singly and doubly glycosylated peptides, respectively. Experimental conditions were optimized to promote the trypsin-mediated (18)O(2) incorporation and prevent backbone exchange. The accuracy, reproducibility, and linearity of relative quantitation were evaluated by using 15 glycoproteins spiked into mouse serum at different concentration ratios. Using this approach, we were able to identify and quantitate 224 N-glycopeptides representing 130 unique glycoproteins from 20 μL of the undepleted mouse serum samples. The strategy can be easily adapted to the analysis of glycoproteins in tissues, cell lines, and other sample origins.

The mammalian target of rapamycin (mTOR) regulates growth via promoting translation and transcription. Here, employing an mTOR active-site inhibitor WYE-125132 (WYE-132), we have performed quantitative phospho-proteomics and identified a Ser-75-containing phosphopeptide from Maf1, a known repressor of RNA polymerase III (Pol III) transcription. Treatment of cancer cells with WYE-132 or the rapamycin analog CCI-779 led to a rapid loss of the phosphorylation at Ser-75, whereas this effect was not seen in cells treated with cytotoxic agents or unrelated inhibitors. WYE-132-induced Maf1 dephosphorylation correlated with its accumulation in the nucleus and a marked decline in the cellular levels of pre-tRNAs. Depletion of cellular Maf1 via small interfering RNA increased basal pre-tRNA and rendered tRNA synthesis refractory to mTOR inhibitors. Maf1 mutant proteins carrying S75A alone or with S60A, T64A, and S68A (Maf1-S75A, Maf1–4A) progressively enhanced basal repression of tRNA in actively proliferating cells and attenuated amino acid-induced tRNA transcription. Gene alignment revealed conservation of all four Ser/Thr sites in high eukaryotes, further supporting a critical role of these residues in Maf1 function. Interestingly, mTOR inhibition led to an increase in the occupancy of Maf1 on a set of Pol III-dependent genes, with concomitant reduction in the binding of Pol III and Brf1. Unexpectedly, mTORC1 itself was also enriched at the same set of Pol III templates, but this association was not influenced by mTOR inhibitor treatment. Our results highlight a new and unique mode of regulation of Pol III transcription by mTOR and suggest that normalization of Pol III activity may contribute to the therapeutic efficacy of mTOR inhibitors.