Brian L. Frey

Discovery and Mass Spectrometric Analysis of Novel Splice-junction Peptides Using RNA-Seq

Human proteomic databases required for MS peptide identification are frequently updated and carefully curated, yet are still incomplete because it has been challenging to acquire every protein sequence from the diverse assemblage of proteoforms expressed in every tissue and cell type. In particular, alternative splicing has been shown to be a major source of this cell-specific proteomic variation. Many new alternative splice forms have been detected at the transcript level using next generation sequencing methods, especially RNA-Seq, but it is not known how many of these transcripts are being translated. Leveraging the unprecedented capabilities of next generation sequencing methods, we collected RNA-Seq and proteomics data from the same cell population (Jurkat cells) and created a bioinformatics pipeline that builds customized databases for the discovery of novel splice-junction peptides. Eighty million paired-end Illumina reads and ∼500,000 tandem mass spectra were used to identify 12,873 transcripts (19,320 including isoforms) and 6810 proteins. We developed a bioinformatics workflow to retrieve high-confidence, novel splice junction sequences from the RNA data, translate these sequences into the analogous polypeptide sequence, and create a customized splice junction database for MS searching. Based on the RefSeq gene models, we detected 136,123 annotated and 144,818 unannotated transcript junctions. Of those, 24,834 unannotated junctions passed various quality filters (e.g. minimum read depth) and these entries were translated into 33,589 polypeptide sequences and used for database searching. We discovered 57 splice junction peptides not present in the Uniprot-Trembl proteomic database comprising an array of different splicing events, including skipped exons, alternative donors and acceptors, and noncanonical transcriptional start sites. To our knowledge this is the first example of using sample-specific RNA-Seq data to create a splice-junction database and discover new peptides resulting from alternative splicing.

Formaldehyde crosslinking: a tool for the study of chromatin complexes

Formaldehyde has been used for decades to probe macromolecular structure and function and to trap complexes, cells, and tissues for further analysis. Formaldehyde crosslinking is routinely employed for detection and quantification of protein-DNA interactions, interactions between chromatin proteins, and interactions between distal segments of the chromatin fiber. Despite widespread use and a rich biochemical literature, important aspects of formaldehyde behavior in cells have not been well described. Here, we highlight features of formaldehyde chemistry relevant to its use in analyses of chromatin complexes, focusing on how its properties may influence studies of chromatin structure and function.

Vibrational spectroscopic studies of the attachment chemistry for zirconium phosphonate multilayers at gold and germanium surfaces

The attachment chemistry required for the sequential self-assembly of zirconium phosphonate (ZP) monolayers on gold and germanium surfaces is examined with a combination of attenuated total reflection (ATR-FTIR) and polarization modulation (PM-FTIR) Fourier transform infrared spectroscopies. On the germanium substrates, the conversion of an attached o-aminosilane monolayer to phosphate amide species creates a primer for the sequential deposition of multilayer films of zirconium 1,lO-decanediylbis(phosphonate) (Zr/DBP). On vapor-depoeited gold f i , a packed monolayer of 11-mercapto-1-undecanol (MUD) serves as the basis for a primer monolayer. PM-FTIR measurements show that phosphorylation of the terminal hydroxy groups results in a surface with a mixture of phosphate monoand diesters, which bind W+ to commence the self-assembly of the Zr/DBP multilayers. On both substrates, the alkyl chain regions of the ZP film are observed to be conformationally disordered due to the control of the lateral spacing in the f i by the inorganic (phoephonate-Zr-phoephonate) regions. Furthermore, the packing density, order, and chemical structure of the primer layer are found to influence the resulting ZP multilayer structure.

Large-scale mass spectrometric detection of variant peptides resulting from nonsynonymous nucleotide differences.

Each individual carries thousands of nonsynonymous single nucleotide variants (nsSNVs) in their genome, each corresponding to a single amino acid polymorphism (SAP) in the encoded proteins. It is important to be able to directly detect and quantify these variations at the protein level to study post-transcriptional regulation, differential allelic expression, and other important biological processes. However, such variant peptides are not generally detected in standard proteomic analyses due to their absence from the generic databases that are employed for mass spectrometry searching. Here we extend previous work that demonstrated the use of customized SAP databases constructed from sample-matched RNA-Seq data. We collected deep-coverage RNA-Seq data from the Jurkat cell line, compiled the set of nsSNVs that are expressed, used this information to construct a customized SAP database, and searched it against deep-coverage shotgun MS data obtained from the same sample. This approach enabled the detection of 421 SAP peptides mapping to 395 nsSNVs. We compared these peptides to peptides identified from a large generic search database containing all known nsSNVs (dbSNP) and found that more than 70% of the SAP peptides from this dbSNP-derived search were not supported by the RNA-Seq data and thus are likely false positives. Next, we increased the SAP coverage from the RNA-Seq derived database by utilizing multiple protease digestions, thereby increasing variant detection to 695 SAP peptides mapping to 504 nsSNV sites. These detected SAP peptides corresponded to moderate to high abundance transcripts (30+ transcripts per million, TPM). The SAP peptides included 192 allelic pairs; the relative expression levels of the two alleles were evaluated for 51 of those pairs and were found to be comparable in all cases.

Proteomic analysis of naturally-sourced biological scaffolds.

A key challenge to the clinical implementation of decellularized scaffold-based tissue engineering lies in understanding the process of removing cells and immunogenic material from a donor tissue/organ while maintaining the biochemical and biophysical properties of the scaffold that will promote growth of newly seeded cells. Current criteria for evaluating whole organ decellularization are primarily based on nucleic acids, as they are easy to quantify and have been directly correlated to adverse host responses. However, numerous proteins cause immunogenic responses and thus should be measured directly to further understand and quantify the efficacy of decellularization. In addition, there has been increasing appreciation for the role of the various protein components of the extracellular matrix (ECM) in directing cell growth and regulating organ function. We performed in-depth proteomic analysis on four types of biological scaffolds and identified a large number of both remnant cellular and ECM proteins. Measurements of individual protein abundances during the decellularization process revealed significant removal of numerous cellular proteins, but preservation of most structural matrix proteins. The observation that decellularized scaffolds still contain many cellular proteins, although at decreased abundance, indicates that elimination of DNA does not assure adequate removal of all cellular material. Thus, proteomic analysis provides crucial characterization of the decellularization process to create biological scaffolds for future tissue/organ replacement therapies.

Modifying the Charge State Distribution of Proteins in Electrospray Ionization Mass Spectrometry by Chemical Derivatization

Electrospray ionization (ESI) of denatured proteins produces a broad distribution of multiply-charged ions leading to multiple peaks in the mass spectrum. We investigated changes in the positive-mode ESI charge state distribution produced by several chemical modifications of denatured proteins. Capping carboxylic acid groups with neutral functional groups yields little change in charge state distribution compared with unmodified proteins. The results indicate that carboxyl groups do not play a significant role in the positive charging of denatured proteins in ESI. The modification of proteins with additional basic sites or fixed positive charges generates substantially higher charge states, providing evidence that the number of ionizable sites, rather than molecular size and shape, determines ESI charging for denatured proteins. Fixed charge modification also significantly reduces the number of protons acquired by a protein, in that the charge state envelope is not increased by the full number of fixed charges appended. This result demonstrates that Coulombic repulsion between positive charges plays a significant role in determining charge state distribution by affecting the gas-phase basicity of ionizable sites. Addition of fixed-charge moieties to a protein is a useful approach for shifting protein charge state distributions to higher charge states, and with further work, it may help limit the distribution of protein ions to fewer charge states.

Using Galaxy-P to leverage RNA-Seq for the discovery of novel protein variations

BackgroundCurrent practice in mass spectrometry (MS)-based proteomics is to identify peptides by comparison of experimental mass spectra with theoretical mass spectra derived from a reference protein database; however, this strategy necessarily fails to detect peptide and protein sequences that are absent from the database. We and others have recently shown that customized proteomic databases derived from RNA-Seq data can be employed for MS-searching to both improve MS analysis and identify novel peptides. While this general strategy constitutes a significant advance for the discovery of novel protein variations, it has not been readily transferable to other laboratories due to the need for many specialized software tools. To address this problem, we have implemented readily accessible, modifiable, and extensible workflows within Galaxy-P, short for Galaxy for Proteomics, a web-based bioinformatic extension of the Galaxy framework for the analysis of multi-omics (e.g. genomics, transcriptomics, proteomics) data.ResultsWe present three bioinformatic workflows that allow the user to upload raw RNA sequencing reads and convert the data into high-quality customized proteomic databases suitable for MS searching. We show the utility of these workflows on human and mouse samples, identifying 544 peptides containing single amino acid polymorphisms (SAPs) and 187 peptides corresponding to unannotated splice junction peptides, correlating protein and transcript expression levels, and providing the option to incorporate transcript abundance measures within the MS database search process (reduced databases, incorporation of transcript abundance for protein identification score calculations, etc.).ConclusionsUsing RNA-Seq data to enhance MS analysis is a promising strategy to discover novel peptides specific to a sample and, more generally, to improve proteomics results. The main bottleneck for widespread adoption of this strategy has been the lack of easily used and modifiable computational tools. We provide a solution to this problem by introducing a set of workflows within the Galaxy-P framework that converts raw RNA-Seq data into customized proteomic databases.

Alkylating tryptic peptides to enhance electrospray ionization mass spectrometry analysis.

A major limitation of mass spectrometry-based proteomics is inefficient and differential ionization during electrospray ionization (ESI). This leads to problems such as increased limits of detection and incomplete sequence coverage of proteins. Incomplete sequence coverage is especially problematic for analyses that require the detection and identification of specific peptides from a protein, such as the analysis of post-translational modifications. We describe here the development and use of aldehyde-based chemistry for the alkylation of peptide primary amines to increase peptide hydrophobicity, providing increased ionization efficiency and concomitant signal enhancement. When employed to modify the peptide products of protein tryptic digests, increased sequence coverage is obtained from combined modified and unmodified digests. To evaluate the utility of alkylation of peptides for selected reaction monitoring (SRM) assays, we alkylated a peptide from the protein Oct4, known to play a role in regulating stem cell differentiation. Increased chromatographic retention and ionization efficiency is observed for the alkylated Oct4 peptide compared to its unmodified form.

Sequence-Specific Capture of Protein-DNA Complexes for Mass Spectrometric Protein Identification

The regulation of gene transcription is fundamental to the existence of complex multicellular organisms such as humans. Although it is widely recognized that much of gene regulation is controlled by gene-specific protein-DNA interactions, there presently exists little in the way of tools to identify proteins that interact with the genome at locations of interest. We have developed a novel strategy to address this problem, which we refer to as GENECAPP, for Global ExoNuclease-based Enrichment of Chromatin-Associated Proteins for Proteomics. In this approach, formaldehyde cross-linking is employed to covalently link DNA to its associated proteins; subsequent fragmentation of the DNA, followed by exonuclease digestion, produces a single-stranded region of the DNA that enables sequence-specific hybridization capture of the protein-DNA complex on a solid support. Mass spectrometric (MS) analysis of the captured proteins is then used for their identification and/or quantification. We show here the development and optimization of GENECAPP for an in vitro model system, comprised of the murine insulin-like growth factor-binding protein 1 (IGFBP1) promoter region and FoxO1, a member of the forkhead rhabdomyosarcoma (FoxO) subfamily of transcription factors, which binds specifically to the IGFBP1 promoter. This novel strategy provides a powerful tool for studies of protein-DNA and protein-protein interactions.

Bioengineered vocal fold mucosa for voice restoration

Primary human vocal fold mucosal cells recapitulate native physiologic function, offering voice restoration to patients with advanced laryngeal disease. Getting vocal about tissue engineering The power of the voice cannot be disputed. For instance, Adele’s lyrics would not elicit chills (or tears) without strategic pitch and harmonizing known as appoggiatura; the chant “Yes we can” garnered more than 69 million popular votes to win Obama the 2008 presidential election; and, more simply, voice is the primary means we all use to communicate with co-workers, loved ones, and the rest of society. Dysphonia—or difficulty speaking from vocal fold tissue damage or loss—can impair one’s ability to be an effective communicator. To provide a new option for those with dysphonia, Ling et al. used two different types of human vocal fold cells to create a functional mucosa. When grafted into the dog larynx ex vivo, the engineered vocal fold reproduced natural physiology, including the vibrations necessary to transmit sound. In vivo, in humanized mice, the engineered mucosa was tolerated by functional human immune cells. These data suggest feasibility for transplant and survival in the larynx as well as for function, ultimately giving patients back their voices. Patients with voice impairment caused by advanced vocal fold (VF) fibrosis or tissue loss have few treatment options. A transplantable, bioengineered VF mucosa would address the individual and societal costs of voice-related communication loss. Such a tissue must be biomechanically capable of aerodynamic-to-acoustic energy transfer and high-frequency vibration and physiologically capable of maintaining a barrier against the airway lumen. We isolated primary human VF fibroblasts and epithelial cells and cocultured them under organotypic conditions. The resulting engineered mucosae showed morphologic features of native tissue, proteome-level evidence of mucosal morphogenesis and emerging extracellular matrix complexity, and rudimentary barrier function in vitro. When grafted into canine larynges ex vivo, the mucosae generated vibratory behavior and acoustic output that were indistinguishable from those of native VF tissue. When grafted into humanized mice in vivo, the mucosae survived and were well tolerated by the human adaptive immune system. This tissue engineering approach has the potential to restore voice function in patients with otherwise untreatable VF mucosal disease.