Many top-down proteomics experiments focus on identifying and localizing post-translational modifications and other potential sources of "mass shift" on a known protein sequence. A simple application to match ion masses and facilitate the iterative hypothesis testing of PTM presence and location would assist with the data analysis in these experiments. ProSight Lite is a free software tool for matching a single candidate sequence against a set of mass spectrometric observations. Fixed or variable modifications, including both post-translational modifications and a select number of glycosylations, can be applied to the amino acid sequence. The application reports multiple scores and a matching fragment list. Fragmentation maps can be exported for publication in either PNG or SVG format. ProSight Lite can be freely downloaded from http:// prosightlite.northwestern.edu, installs and updates from the web, and requires Windows 7 or higher. KeywordsTop-down proteomics; proteomics software; MS Analysis; proteoform characterization Top-down proteomics describes the study of intact proteins with mass spectrometry [1,2]. Traditional bottom-up proteomics experiments are marked by the use of an enzyme, typically trypsin, to proteolyze intact proteins into more analytically manageable peptides (0.5-3 kDa) [3]. This proteolysis effects a loss of information between the ribosomallyexpressed pro-protein, which in eukaryotes often contains RNA splice variants, and the posttranslationally modified intact protein (termed a "proteoform") [4]. If a modification has been identified on two separate peptides, a typical bottom-up proteomics experiment cannot know whether those modification existed singly on two separate proteoforms or in tandem on a single proteoform. While more analytically challenging, top-down proteomics provides that full information [5]. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptAnalysis of top-down mass spectrometry data is separated into two main steps. First, the "analysis to infer mass (AIM)" step takes the complex m/z data provided by the mass spectrometer, performs deconvolution and deisotoping to provide neutral (or singly-charged) masses [6]. Second, those masses (with settings informed by experimental metadata) are matched against a hypothetical proteoform to determine the degree of matching. In this manuscript, we describe the release of ProSight Lite, a freely available application for targeted top-down proteomics data analysis. In a targeted top-down experiment, the user has generated a hypothesis (or set of hypotheses) for the identity of the proteoform under study (either through their experimental design or other, more complex 'discovery' experiments). The goal of a targeted top-down experiment is often to identify and localize posttranslational modifications (PTMs) on a known protein sequence. Offered as a partial replacement for our group's previously described software, ProSightPTM [7] and ProSightPTM2 [8], ProSight Lite describes a simple, i...

Formalin-fixed, paraffin-embedded (FFPE), biobanked tissue samples offer an invaluable resource for clinical and biomarker research. Here, we developed a pressure cycling technology (PCT)-SWATH mass spectrometry workflow to analyze FFPE tissue proteomes and applied it to the stratification of prostate cancer (PCa) and diffuse large B-cell lymphoma (DLBCL) samples. We show that the proteome patterns of FFPE PCa tissue samples and their analogous fresh-frozen (FF) counterparts have a high degree of similarity and we confirmed multiple proteins consistently regulated in PCa tissues in an independent sample cohort. We further demonstrate temporal stability of proteome patterns from FFPE samples that were stored Abbreviations BPH, benign prostatic hyperplasia; CRYAB, crystallin alpha Bbetween 1 and 15 years in a biobank and show a high degree of the proteome pattern similarity between two types of histological regions in small FFPE samples, that is, punched tissue biopsies and thin tissue sections of micrometer thickness, despite the existence of a certain degree of biological variations. Applying the method to two independent DLBCL cohorts, we identified myeloperoxidase, a peroxidase enzyme, as a novel prognostic marker. In summary, this study presents a robust proteomic method to analyze bulk and biopsy FFPE tissues and reports the first systematic comparison of proteome maps generated from FFPE and FF samples. Our data demonstrate the practicality and superiority of FFPE over FF samples for proteome in biomarker discovery. Promising biomarker candidates for PCa and DLBCL have been discovered.

Cell membrane-covered drug-delivery nanoplatforms have been garnering attention because of their enhanced biointerfacing capabilities that originate from source cells. In this top-down technique, nanoparticles (NPs) are covered by various membrane coatings, including membranes from specialized cells or hybrid membranes that combine the capacities of different types of cell membranes. Here, hybrid membrane-coated doxorubicin (Dox)-loaded poly(lacticco-glycolic acid) (PLGA) NPs (DPLGA@[RAW-4T1] NPs) were fabricated by fusing membrane components derived from RAW264.7(RAW) and 4T1 cells (4T1). These NPs were used to treat lung metastases originating from breast cancer. This study indicates that the coupling of NPs with a hybrid membrane derived from macrophage and cancer cells has several advantages, such as the tendency to accumulate at sites of inflammation, ability to target specific metastasis, homogenous tumor targeting abilities in vitro, and markedly enhanced multi-target capability in a lung metastasis model in vivo. The DPLGA@[RAW-4T1] NPs exhibited excellent chemotherapeutic potential with approximately 88.9% anti-metastasis efficacy following treatment of breast cancer-derived lung metastases. These NPs were robust and displayed the multi-targeting abilities of hybrid membranes. This study provides a promising biomimetic nanoplatform for effective treatment of breast cancer metastasis.

The effects of varying the electron energy and cationizing agents on electron activated dissociation (ExD) of metal-adducted oligosaccharides were explored, using permethylated maltoheptaose as the model system. Across the examined range of electron energy, the metal-adducted oligosaccharide exhibited several fragmentation processes, including electron capture dissociation (ECD) at low energies, hot-ECD at intermediate energies, and electronic excitation dissociation (EED) at high energies. The dissociation threshold depended on the metal charge carrier(s), whereas the types and sequence spans of product ions were influenced by the metal-oligosaccharide binding pattern. Theoretical modeling contributed insight into the metal-dependent behavior of carbohydrates during low-energy ECD. When ExD was applied to a permethylated high mannose N-linked glycan, EED provided more structural information than either collision-induced dissociation (CID) or low-energy ECD, thus demonstrating its potential for oligosaccharide linkage analysis.

The structural complexity and diversity of glycans parallel their multilateral functions in living systems. To better understand the vital roles glycans play in biological processes, it is imperative to develop analytical tools that can provide detailed glycan structural information. This was conventionally achieved by multistage tandem mass spectrometry (MSn) analysis using collision-induced dissociation (CID) as the fragmentation method. However, the MSn approach lacks the sensitivity and throughput needed to analyze complex glycan mixtures from biological sources, often available in limited quantities. We define herein the critical parameters for a recently developed fragmentation technique, electronic excitation dissociation (EED), which can yield rich structurally informative fragment ions during liquid chromatographic (LC)-MS/MS analysis of glycans. We further demonstrate that permethylation, reducing end labeling and judicious selection of the metal charge carrier can greatly facilitate spectral interpretation. With its high sensitivity, throughput, and compatibility with on-line chromatographic separation techniques, EED appears to hold great promise for large-scale glycomics studies.

A modular and adaptive mass spectrometry (MS)-based platform was developed to provide fast, robust and sensitive host cell protein (HCP) analytics to support process development. This platform relies on one-dimensional ultra-high performance liquid chromatography (1D UHPLC) combined with several different MS data acquisition strategies to meet the needs of purification process development. The workflow was designed to allow HCP composition and quantitation for up to 20 samples per day, a throughput considered essential for real time bioprocess development support. With data-dependent acquisition (DDA), the 1D UHPLC-MS/MS method had excellent speed and demonstrated robustness in detecting unknown HCPs at 50 ng/mg (ppm) level. Combining 1D UHPLC with sequential window acquisition of all theoretical spectra (SWATH) MS enabled simultaneous detection and quantitation of all HCPs in single-digit ng/mg range within 1 hour, demonstrating for the first time the benefit of SWATH MS as a technique for HCP analysis. As another alternative, a targeted MS approach can be used to track the clearance of specific known HCP under various process conditions. This study highlights the importance of designing a robust LC-MS/MS workflow that not only allows HCP discovery, but also affords greatly improved process knowledge and capability in HCP removal. As an orthogonal and complementary detection approach to traditional HCP analysis by enzyme-linked immunosorbent assay, the reported LC-MS/MS workflow supports the development of bioprocesses with optimal HCP clearance and the production of safe and high quality therapeutic biopharmaceuticals.

Translation errors in protein biosynthesis may result in low level amino acid misincorporation and contribute to product heterogeneity of recombinant protein therapeutics. We report the use of peptide map analysis by reversed-phase high-performance liquid chromatography and high-resolution mass spectrometry to detect and identify mistranslation events in recombinant monoclonal antibodies expressed in mammalian cell lines including Chinese hamster ovary (CHO) cells. Misincorporation of an asparagine residue at multiple serine positions was detected as earlier-eluting peptides with masses 27.01 Da higher than expected. The exact positions at which misincorporation occurred were identified by tandem mass spectrometry of the asparagine-containing variant peptides. The identified asparagine misincorporation sites correlated with the use of codon AGC but with none of the other five serine codons. The relative levels of misincorporation ranged from 0.01%-0.2% among multiple serine positions detected across three different antibodies by targeted analysis of expected and variant peptides. The low levels of misincorporation are consistent with published predictions for in vivo translation error rates. Our results demonstrate that state-of-the-art mass spectrometry with a combination of high sensitivity, accuracy, and dynamic range provides a new ability to discover and characterize low level protein variants that arise from mistranslation events.

The sites of glycosylation of Chinese hamster ovary cell expressed testicular angiotensin-converting enzyme (tACE) have been determined by matrix-assisted laser desorption ionization/time of flight/mass spectrometry of peptides generated by proteolytic and cyanogen bromide digestion. Two of the seven potential Nlinked glycosylation sites, Asn 90 and Asn 109 , were found to be fully glycosylated by analysis of peptides before and after treatment with a series of glycosidases and with endoproteinase Asp-N. The mass spectra of the glycopeptides exhibit characteristic clusters of peaks which indicate the N-linked glycans in tACE to be mostly of the biantennary, fucosylated complex type. This structural information was used to demonstrate that three other sites, Asn 155 , Asn 337 , and Asn 586 , are partially glycosylated, whereas Asn 72 appears to be fully glycosylated. The only potential site that was not modified is Asn 620 . Sequence analysis of tryptic peptides obtained from somatic ACE (human kidney) identified six glycosylated and one unglycosylated Asn. Only one of these glycosylation sites had a counterpart in tACE. Comparison of the two proteins reveals a pattern in which amino-terminal N-linked sites are preferred. The functional significance of glycosylation was examined with a tACE mutant lacking the O-glycan-rich first amino-terminal 36 residues and truncated at Ser 625 . When expressed in the presence of the ␣-glucosidase I inhibitor N-butyldeoxynojirimycin and treated with endoglycosidase H to remove all but the terminal N-acetylglucosamine residues, it retained full enzymatic activity, was electrophoretically homogeneous, and is a good candidate for crystallographic studies.Both forms of angiotensin-converting enzyme (ACE 1 ; EC 3.4.15.1 peptidyl-dipeptidase A) are class I transmembrane ectoenzymes (1) that have N-and O-linked oligosaccharides attached to their polypeptide chains (2, 3). Expression of ACE in human HeLa cells in the presence of tunicamycin resulted in complete inhibition of glycosylation, rapidly degraded intracellular ACE, and no enzyme released in the medium (4). An enzymatically active ACE was produced with partial glycosylation in a mutant Chinese hamster ovary (CHO) cell line (ldlD), although it was released to a lesser extent (4). Similarly, it was reported (5) that inhibitors of glucosidases I and II in the endoplasmic reticulum (ER) and mannosidase I in the cis-Golgi reduced the amount of oligosaccharide attached to human intestinal ACE and delayed protein release significantly. These data strongly suggest that glycosylation plays an important role in the membrane targeting and release of ACE, possibly by affecting the folding of the polypeptide and its recognition by a variety of enzymes in the folding and transport machineries. Recently, Sadhukhan and Sen (6) reported that mutations at individual N-linked glycosylation sites (sequons) in rabbit testis ACE (tACE) resulted in varied efficiencies in enzyme release, which suggests that N-linked glycans at each site may make diff...