Matthew Fitzgibbon

Quantitative analysis of acrylamide labeled serum proteins by LC-MS/MS

Isotopic labeling of cysteine residues with acrylamide was previously utilized for relative quantitation of proteins by MALDI-TOF. Here, we explored and compared the application of deuterated and (13)C isotopes of acrylamide for quantitative proteomic analysis using LC-MS/MS and high-resolution FTICR mass spectrometry. The method was applied to human serum samples that were immunodepleted of abundant proteins. Our results show reliable quantitation of proteins across an abundance range that spans 5 orders of magnitude based on ion intensities and known protein concentration in plasma. The use of (13)C isotope of acrylamide had a slightly greater advantage relative to deuterated acrylamide, because of shifts in elution of deuterated acrylamide relative to its corresponding nondeuterated compound by reversed-phase chromatography. Overall, the use of acrylamide for differentially labeling intact proteins in complex mixtures, in combination with LC-MS/MS provides a robust method for quantitative analysis of complex proteomes.

Myc-dependent mitochondrial generation of acetyl-CoA contributes to fatty acid biosynthesis and histone acetylation during cell cycle entry.

Cell reprogramming from a quiescent to proliferative state requires coordinate activation of multiple -omic networks. These networks activate histones, increase cellular bioenergetics and the synthesis of macromolecules required for cell proliferation. However, mechanisms that coordinate the regulation of these interconnected networks are not fully understood. The oncogene c-Myc (Myc) activates cellular metabolism and global chromatin remodeling. Here we tested for an interconnection between Myc regulation of metabolism and acetylation of histones. Using [13C6]glucose and a combination of GC/MS and LC/ESI tandem mass spectrometry, we determined the fractional incorporation of 13C-labeled 2-carbon fragments into the fatty acid palmitate, and acetyl-lysines at the N-terminal tail of histone H4 in myc−/− and myc+/+ Rat1A fibroblasts. Our data demonstrate that Myc increases mitochondrial synthesis of acetyl-CoA, as the de novo synthesis of 13C-labeled palmitate was increased 2-fold in Myc-expressing cells. Additionally, Myc induced a forty percent increase in 13C-labeled acetyl-CoA on H4-K16. This is linked to the capacity of Myc to increase mitochondrial production of acetyl-CoA, as we show that mitochondria provide 50% of the acetyl groups on H4-K16. These data point to a key role for Myc in directing the interconnection of -omic networks, and in particular, epigenetic modification of proteins in response to proliferative signals.

Plasma proteome profiling of a mouse model of breast cancer identifies a set of up-regulated proteins in common with human breast cancer cells.

We have applied an in-depth quantitative proteomic approach, combining isotopic labeling extensive intact protein separation and mass spectrometry, for high confidence identification of protein changes in plasmas from a mouse model of breast cancer. We hypothesized that a wide spectrum of proteins may be up-regulated in plasma with tumor development and that comparisons with proteins expressed in human breast cancer cell lines may identify a subset of up-regulated proteins in common with proteins expressed in breast cancer cell lines that may represent candidate biomarkers for breast cancer. Plasma from PyMT transgenic tumor-bearing mice and matched controls were obtained at two time points during tumor growth. A total of 133 proteins were found to be increased by 1.5-fold or greater at one or both time points. A comparison of this set of proteins with published findings from proteomic analysis of human breast cancer cell lines yielded 49 proteins with increased levels in mouse plasma that were identified in breast cancer cell lines. Pathway analysis comparing the subset of up-regulated proteins known to be expressed in breast cancer cell lines with other up-regulated proteins indicated a cancer related function for the former and a host-response function for the latter. We conclude that integration of proteomic findings from mouse models of breast cancer and from human breast cancer cell lines may help identify a subset of proteins released by breast cancer cells into the circulation and that occur at increased levels in breast cancer.

Brain-specific Proteins Decline in the Cerebrospinal Fluid of Humans with Huntington Disease

We integrated five sets of proteomics data profiling the constituents of cerebrospinal fluid (CSF) derived from Huntington disease (HD)-affected and -unaffected individuals with genomics data profiling various human and mouse tissues, including the human HD brain. Based on an integrated analysis, we found that brain-specific proteins are 1.8 times more likely to be observed in CSF than in plasma, that brain-specific proteins tend to decrease in HD CSF compared with unaffected CSF, and that 81% of brain-specific proteins have quantitative changes concordant with transcriptional changes identified in different regions of HD brain. The proteins found to increase in HD CSF tend to be liver-associated. These protein changes are consistent with neurodegeneration, microgliosis, and astrocytosis known to occur in HD. We also discuss concordance between laboratories and find that ratios of individual proteins can vary greatly, but the overall trends with respect to brain or liver specificity were consistent. Concordance is highest between the two laboratories observing the largest numbers of proteins.

Identification of long-lived proteins retained in cells undergoing repeated asymmetric divisions

Significance Long-lived proteins in extracellular spaces (joints/tissues) or within specialized nondividing cells (eye-lens) are associated with age-related decline. However, aging also occurs in dividing stem cells. Although several hypotheses have been proposed to explain how stem cells age, none have addressed whether long-lived proteins contribute to aging, partially because of technical challenges in identifying such proteins. We developed a method to overcome these limitations in the model system Saccharomyces cerevisiae. We identified two classes of long-lived asymmetrically retained proteins (LARPs). Full-length LARPs remain intact throughout the mother cell lifespan and accumulate in abundance or become posttranslationally modified. Fragmented LARPs are original proteins that are partially degraded, yet retained by the mother cell during aging. We speculate that LARPs contribute to the aging process. Long-lived proteins have been implicated in age-associated decline in metazoa, but they have only been identified in extracellular matrices or postmitotic cells. However, the aging process also occurs in dividing cells undergoing repeated asymmetric divisions. It was not clear whether long-lived proteins exist in asymmetrically dividing cells or whether they are involved in aging. Here we identify long-lived proteins in dividing cells during aging using the budding yeast, Saccharomyces cerevisiae. Yeast mother cells undergo a limited number of asymmetric divisions that define replicative lifespan. We used stable-isotope pulse-chase and total proteome mass-spectrometry to identify proteins that were both long-lived and retained in aging mother cells after ∼18 cells divisions. We identified ∼135 proteins that we designate as long-lived asymmetrically retained proteins (LARPS). Surprisingly, the majority of LARPs appeared to be stable fragments of their original full-length protein. However, 15% of LARPs were full-length proteins and we confirmed several candidates to be long-lived and retained in mother cells by time-lapse microscopy. Some LARPs localized to the plasma membrane and remained robustly in the mother cell upon cell division. Other full-length LARPs were assembled into large cytoplasmic structures that had a strong bias to remain in mother cells. We identified age-associated changes to LARPs that include an increase in their levels during aging because of their continued synthesis, which is not balanced by turnover. Additionally, several LARPs were posttranslationally modified during aging. We suggest that LARPs contribute to age-associated phenotypes and likely exist in other organisms.

Integrative Proteomic Analysis of Serum and Peritoneal Fluids Helps Identify Proteins that Are Up-Regulated in Serum of Women with Ovarian Cancer

Background We used intensive modern proteomics approaches to identify predictive proteins in ovary cancer. We identify up-regulated proteins in both serum and peritoneal fluid. To evaluate the overall performance of the approach we track the behavior of 20 validated markers across these experiments. Methodology Mass spectrometry based quantitative proteomics following extensive protein fractionation was used to compare serum of women with serous ovarian cancer to healthy women and women with benign ovarian tumors. Quantitation was achieved by isotopically labeling cysteine amino acids. Label-free mass spectrometry was used to compare peritoneal fluid taken from women with serous ovarian cancer and those with benign tumors. All data were integrated and annotated based on whether the proteins have been previously validated using antibody-based assays. Findings We selected 54 quantified serum proteins and 358 peritoneal fluid proteins whose case-control differences exceeded a predefined threshold. Seventeen proteins were quantified in both materials and 14 are extracellular. Of 19 validated markers that were identified all were found in cancer peritoneal fluid and a subset of 7 were quantified in serum, with one of these proteins, IGFBP1, newly validated here. Conclusion Proteome profiling applied to symptomatic ovarian cancer cases identifies a large number of up-regulated serum proteins, many of which are or have been confirmed by immunoassays. The number of currently known validated markers is highest in peritoneal fluid, but they make up a higher percentage of the proteins observed in both serum and peritoneal fluid, suggesting that the 10 additional markers in this group may be high quality candidates.

Biomarker validation by targeted mass spectrometry

A multilaboratory study demonstrates the potential for establishing quantitative targeted proteomic assays for moderately to highly abundant plasma proteins.

Tumor-infiltrating merkel cell polyomavirus-specific T cells are diverse and associated with improved patient survival

Merkel cell carcinoma patients had improved survival if their tumors contained a greater frequency or diversity of T cells that were specific for a prevalent Merkel cell polyomavirus epitope. Thus, transgenic T-cell receptor therapy could potentially benefit patients. Tumor-infiltrating CD8+ T cells are associated with improved survival of patients with Merkel cell carcinoma (MCC), an aggressive skin cancer causally linked to Merkel cell polyomavirus (MCPyV). However, CD8+ T-cell infiltration is robust in only 4% to 18% of MCC tumors. We characterized the T-cell receptor (TCR) repertoire restricted to one prominent epitope of MCPyV (KLLEIAPNC, “KLL”) and assessed whether TCR diversity, tumor infiltration, or T-cell avidity correlated with clinical outcome. HLA-A*02:01/KLL tetramer+ CD8+ T cells from MCC patient peripheral blood mononuclear cells (PBMC) and tumor-infiltrating lymphocytes (TIL) were isolated via flow cytometry. TCRβ (TRB) sequencing was performed on tetramer+ cells from PBMCs or TILs (n = 14) and matched tumors (n = 12). Functional avidity of T-cell clones was determined by IFNγ production. We identified KLL tetramer+ T cells in 14% of PBMC and 21% of TIL from MCC patients. TRB repertoires were strikingly diverse (397 unique TRBs were identified from 12 patients) and mostly private (only one TCRb clonotype shared between two patients). An increased fraction of KLL-specific TIL (>1.9%) was associated with significantly increased MCC-specific survival P = 0.0009). T-cell cloning from four patients identified 42 distinct KLL-specific TCRa/b pairs. T-cell clones from patients with improved MCC-specific outcomes were more avid (P < 0.05) and recognized an HLA-appropriate MCC cell line. T cells specific for a single MCPyV epitope display marked TCR diversity within and between patients. Intratumoral infiltration by MCPyV-specific T cells was associated with significantly improved MCC-specific survival, suggesting that augmenting the number or avidity of virus-specific T cells may have therapeutic benefit. Cancer Immunol Res; 5(2); 137–47. ©2017 AACR.

Phenotypic and Transcriptional Fidelity of Patient-Derived Colon Cancer Xenografts in Immune-Deficient Mice

Xenografts of human colorectal cancer (CRC) in immune-deficient mice have great potential for accelerating the study of tumor biology and therapy. We evaluated xenografts established in NOD/scid/IL2Rγ-null mice from the primary or metastatic tumors of 27 patients with CRC to estimate their capacity for expanding tumor cells for in vitro studies and to assess how faithfully they recapitulated the transcriptional profile of their parental tumors. RNA-seq analysis of parental human CRC tumors and their derivative xenografts demonstrated that reproducible transcriptional changes characterize the human tumor to murine xenograft transition. In most but not all cases, the human stroma, vasculature, and hematopoietic elements were systematically replaced by murine analogues while the carcinoma component persisted. Once established as xenografts, human CRC cells that could be propagated by serial transplantation remained transcriptionally stable. Three histologically atypical xenografts, established from patients with peritoneal metastases, contained abundant human stromal elements and blood vessels in addition to human tumor cells. The transcriptomes of these mixed tumor/stromal xenografts did not closely resemble those of their parental tumors, and attempts to propagate such xenografts by serial transplantation were unsuccessful. Stable expression of numerous genes previously identified as high priority targets for immunotherapy was observed in most xenograft lineages. Aberrant expression in CRC cells of human genes that are normally only expressed in hematopoietic cells was also observed. Our results suggest that human CRC cells expanded in murine xenografts have great utility for studies of tumor immunobiology and targeted therapies such as immunotherapy but also identify potential limitations.

Worldwide circulation of HSV-2 × HSV-1 recombinant strains

Homo sapiens harbor two distinct, medically significant species of simplexviruses, herpes simplex virus (HSV)-1 and HSV-2, with estimated divergence 6–8 million years ago (MYA). Unexpectedly, we found that circulating HSV-2 strains can contain HSV-1 DNA segments in three distinct genes. Using over 150 genital swabs from North and South America and Africa, we detected recombinants worldwide. Common, widely distributed gene UL39 genotypes are parsimoniously explained by an initial >457 basepair (bp) HSV-1 × HSV-2 crossover followed by back-recombination to HSV-2. Blocks of >244 and >539 bp of HSV-1 DNA within genes UL29 and UL30, respectively, have reached near fixation, with a minority of strains retaining sequences we posit as ancestral HSV-2. Our data add to previous in vitro and animal work, implying that in vivo cellular co-infection with HSV-1 and HSV-2 yields viable interspecies recombinants in the natural human host.