Detection of Differentially Expressed Basal Cell Proteins by Mass Spectrometry

The ability of cells to modulate interactions with each other and the substrate is essential for epithelial tissue remodeling during processes such as wound healing and tumor progression. However, despite strides made in the field of proteomics, proteins involved in adhesion have been difficult to study. Here, we report a method for the enrichment and analysis of proteins associated with the basal surface of the cell and its underlying matrix. The enrichment involves deroofing the cells with 20 mm ammonium hydroxide and the removal of cytosolic and organellar proteins by stringent water wash. Proteomic profiling was achieved by LC-FTMS, which allowed comparison of differentially expressed or shared proteins under different cell states. First, we analyzed and compared the basal cell components of mouse keratinocytes lacking the cell-cell junction molecule plakoglobin with their control counterparts. Changes in the molecules involved in motility and invasion were detected in plakoglobin-deficient cells, including decreased detection of fibronectin, integrin β₄, and FAT tumor suppressor. Second, we assessed the differences in basal cell components between two human oral squamous cell carcinoma lines originating from different sites in the oral cavity (CAL33 and UM-SCC-1). The data show differences between the two lines in the type and abundance of proteins specific to cell adhesion, migration, and angiogenesis. Therefore, the method described here has the potential to serve as a platform to assess proteomic changes in basal cell components including extracellular and adhesion-specific proteins involved in wound healing, cancer, and chronic and acquired adhesion-related disorders.There is an urgent need for tools to comprehensively identify markers of normal and pathological processes at the molecular level. DNA microarrays have enabled researchers to follow gene expression changes with respect to many of these processes, including individual tumors in the case of cancer (1). Direct detection of proteins is typically required to validate changes at the gene product level; however, the changes in protein levels do not always reflect changes in gene expression because of post-translational modifications, differential compartmentalization, recycling, and degradation. Because it is ultimately the proteins that convey cellular phenotypes, it is necessary to develop methods for direct screening of proteins, and mass spectrometry shows promise for this purpose. However, the usefulness of mass spectrometry as an analytical tool to detect proteins in cells or tissue is limited to the extent to which the sample is sufficiently enriched for the specific fraction of interest. It is still challenging to identify molecules involved in specific normal or pathological processes because the relevant proteins are often difficult to isolate from the majority of cellular proteins that are not correlated to the process of interest. In this context, an ideal proteomics approach would require a minimal amount of starting material, be amenable to an efficient enrichment strategy, and would provide results quickly.It has been well established that molecules directly involved in cell-cell and cell-substrate adhesions are critical for processes such as epithelial to mesenchymal transition and wound healing. Their further role in regulation of tissue integrity, cell polarity, motility, and invasion is emphasized by a variety of disorders stemming from their inappropriate expression and mutations (2, 3). Selectins, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 have been established both as biomarkers (4) and predictive factors (5, 6) for the development of accelerated atherosclerosis and heart disease. In epithelial tissues, reduced expression of the cell-cell adhesion molecule E-cadherin correlates with epithelial to mesenchymal transition, tissue invasion, and metastasis and is a prognostic biomarker of poor clinical outcome in many cell types (7–9). Furthermore, up-regulating E-cadherin is considered as a treatment option in several types of cancer (10). Therefore, methods are also needed to not only identify adhesion molecules as disease markers but to also understand the pathology of underlying medical problems caused by impairment in adhesion molecule function (e.g. inability to heal chronic wounds (11)). However, the lack of knowledge about regulation and functional interactions of the specific adhesion-related proteins has so far thwarted the attempts at direct targeting of these molecules in basic and clinical research (12, 13). Therefore, a comprehensive understanding of how proteins that function in adhesive processes work together to maintain proper tissue form and function is critical.Some of the same barriers to effective application of mass spectrometry as an analytical tool (as discussed above) have impeded analysis of cell-cell and cell-matrix adhesion-dependent processes such as wound healing and cancer (14). The study of extracellular matrix (ECM)1 and adhesion-related proteins is further complicated by the difficulty in sample preparation because compared with cytosolic proteins basal cell proteins are often highly insoluble (e.g. transmembrane and plaque components) and difficult to isolate from intracellular proteins. One general strategy involves using ECM-specific enzymes to dislodge the cells at their points of attachment (15). The supernatant from the partial digest is collected for further proteomics analysis. However, most mass spectrometric analyses depend on detection of peptides with specific ionization and fragmentation properties that are most readily achieved using trypsin as the sole enzyme. The use of ECM-specific enzymes may result in a distribution of peptides that are not optimal for detection (i.e. the generation of non-tryptic termini). The other general approach to isolate components of the ECM involves using detergents to lyse cells on the surfaces to which they are attached and collect the remaining cell debris for analysis (15). Although progress has been made with respect to the creation of “mass spectrometry-friendly” detergents (16), the use of chemicals for the purpose of protein solubilization is generally not ideal. To overcome these problems, we adapted a fast, simple method of isolating extracellular, transmembrane, and associated proteins (from here on collectively referred to as “basal cell proteins”) from cells attached to a solid substrate. The method consists of “deroofing” the cells attached to glass coverslips by 20 mm NH₄OH solution followed by rapid water rinses to remove the bulk of the cell and its remaining debris (17). Our results show efficient removal of cytoplasm and organelles and detection of basal cell proteins by mass spectrometry, including those involved in cell-cell and cell-extracellular matrix interactions. These proteins were liberated from the surface with trypsin, and the subsequently generated peptides were detected and profiled for differences using LC-FTMS.The approach was first validated by comparing basal cell protein composition in mouse keratinocytes with or without a critical cell-cell junction protein called plakoglobin (PG). This desmosomal protein is required for cell-cell adhesion and maintenance of tissue integrity (18). Plakoglobin inhibits keratinocyte motility (19) and is down-regulated in several distinct tumor types, including bladder, breast, and cervical cancers (20–22). Moreover, we were able to dissect the molecular differences between an independent isolate of PG^−/− keratinocytes that behaved differently in motility assays from the rest of the PG-null cells, further emphasizing the potential for using the method to differentiate between cells with distinct adhesive and motile behaviors. The method was then evaluated in clinically relevant human tumor cell lines by extending the analysis to include two human oral squamous cancers of different origin. Because they lack precisely defined changes in cell adhesion molecules and phenotype, we compared the basal cell protein expression of UM-SCC-1 (23) and CAL33 (24) cell lines isolated from the roof of the mouth and tongue, respectively. These experiments revealed 40 proteins differentially expressed between the cell lines among over 100 detected. Moreover, the proteomic profile reveals a set of motility- and invasion-related genes unique to tongue-derived CAL33 cells. This could indicate the difference between oral cancers derived from different parts of the mouth, or it may indicate a potential difference in aggressiveness between these cell lines. These results show that our detection method is applicable for both detection and comparative studies in human cancer model systems.     

小鼠晶体蛋白质组的双向电泳和质谱鉴定研究

目的应用双向电泳和质谱技术研究5周龄小鼠晶体蛋白质组。方法提取小鼠晶体总蛋白,进行固相pH梯度（IPG）等电聚焦双向电泳,胶体考马斯亮蓝R-250染色,使用PDQuest7．30图像分析软件分析电泳图像。选择主要蛋白点胶上酶解,应用基质辅助激光解析电离飞行时间／飞行时间（MALDI—TOF／TOF）仪器进行串联质谱（MS／MS）鉴定。结果上样量为882μg和190μg时,分别检测370±41蛋白点（n=3）和57±5个蛋白点（n=3）。高上样量能够较好地分离晶体低丰度蛋白,如念珠状纤维结构蛋白BFSP;低上样量可很好地分离高丰度蛋白-晶体蛋白（包括αA、αB;βA1～βA4;βB1～βB3;γA～γF和γS等）。质谱鉴定得到1种细胞骨架蛋白和16种高丰度晶体蛋白。结论双向电泳和质谱技术有效考察了晶体总蛋白质,为分析白内障形成过程中蛋白质的表达改变提供了新的方法和途径。     

液质联用多反应监测法定量目标多肽或蛋白质

为建立优化的血浆内源性多肽提取方法,并且构建目标多肽和蛋白质的质谱定量方 法,本研究考察了超滤法、有机溶剂沉淀法和固相萃取法对血浆内源性多肽的提取效果 ,并通过Tricine-SDS-PAGE对提取效果进行比较.通过液相色谱串联质谱多反应监测 （MRM）分析,建立了多肽标准品ESAT-6定量方法,并将ESAT-6定量建立的液相色谱和质谱条件应用于蛋白质的定量,对多肽和蛋白质MRM定量的标准曲线进行了考 察.Tricine-SDS-PAGE结果表明,乙腈沉淀法是最佳的血浆内源性多肽提取方法,低分子量的多肽可以得到很好的富集,且能有效地去除高分子蛋白质的污染.液相色谱串联 质谱MRM法检测血浆内提取的多肽,标准曲线的线性较好,相关系数为0.999.另外,采 用MRM法对胶内分离的蛋白质进行定量,标准曲线的线性相关系数为0.995.综上所述, 本研究构建了一种简单有效的血浆多肽提取方法,通过液质联用MRM法成功地实现了目标多肽和蛋白质定量测定.该定量方法可以推广应用于复杂样品中的多肽和蛋白质的定 量分析.     

Distinguishing Small Molecular Mass Differences of Proteins by Mass Spectrometry

Electrospray ionization–Fourier transform ion cyclotron resonance (ESI–FTICR) mass spectrometryallows for high-resolution, accurate mass analysisof multiply charged ions of proteins. In the workdescribed here, the ability of ESI–FTICR to distinguish small differences in molecular mass is evaluated. Ubiquitin was used as an internal mass calibration standard to measure the molecular mass of cytochromec, myoglobin, and several carbonic anhydrase isoforms. Mass calibration was based onthe tallest isotopic peak of each ubiquitin chargestate. Ubiquitin performed well as an internal standard because its charge states covered the appropriate mass range, interference was minimal, and thetallest peak was easily identified. The peak massesof cytochrome c (12.5 kDa) and myoglobin (17 kDa) were measured to an accuracy of about 0.02 Da (<2ppm). However, errors of 1.0 Da were observedfor some individual determinations because of the difficulty in identifying the tallest peak. When the technique was applied to bovine carbonic anhydrase II, even combining data from several charge statesdid not yield an unequivocal assignment of thetallest peak, resulting in a mass assignment of 29,023.7 or 29,024.7. Similarly, measurements of two isoforms with a mass difference of 1 Da, human carbonic anhydrase I, pI6.0 and 6.6, yielded overlapping values for the mass of the tallest peak. However, these two isoforms were clearly distinguished by (a) identification of the tallest peak using a measurement of average mass as a guide and (b) comparison of the isotopic peak intensity patterns.     

Effective Identification of Akt Interacting Proteins by Two-Step Chemical Crosslinking,Co-Immunoprecipitation and Mass Spectrometry

Akt is a critical protein for cell survival and known to interact with various proteins. However, Akt binding partners that modulate or regulate Akt activation have not been fully elucidated. Identification of Akt-interacting proteins has been customarily achieved by co-immunoprecipitation combined with western blot and/or MS analysis. An intrinsic problem of the method is loss of interacting proteins during procedures to remove non-specific proteins. Moreover, antibody contamination often interferes with the detection of less abundant proteins. Here, we developed a novel two-step chemical crosslinking strategy to overcome these problems which resulted in a dramatic improvement in identifying Akt interacting partners. Akt antibody was first immobilized on protein A/G beads using disuccinimidyl suberate and allowed to bind to cellular Akt along with its interacting proteins. Subsequently, dithiobis[succinimidylpropionate], a cleavable crosslinker, was introduced to produce stable complexes between Akt and binding partners prior to the SDS-PAGE and nanoLC-MS/MS analysis. This approach enabled identification of ten Akt partners from cell lysates containing as low as 1.5 mg proteins, including two new potential Akt interacting partners. None of these but one protein was detectable without crosslinking procedures. The present method provides a sensitive and effective tool to probe Akt-interacting proteins. This strategy should also prove useful for other protein interactions, particularly those involving less abundant or weakly associating partners.     

Rapid Identification of Probiotic Lactobacillus Biosurfactant Proteins by ProteinChip Tandem Mass Spectrometry Tryptic Peptide Sequencing

A novel ProteinChip-interfaced tandem mass spectrometer was employed to identify collagen binding proteins from biosurfactant produced by Lactobacillus fermentum RC-14. On-chip tryptic digestion of the captured collagen binding proteins resulted in rapid sequence identification of five novel tryptic peptide sequences via collision-induced dissociation tandem mass spectrometry.     

Identification and Quantification of Proteoforms by Mass Spectrometry

A proteoform is a defined form of a protein derived from a given gene with a specific amino acid sequence and localized post‐translational modifications. In top‐down proteomic analyses, proteoforms are identified and quantified through mass spectrometric analysis of intact proteins. Recent technological developments have enabled comprehensive proteoform analyses in complex samples, and an increasing number of laboratories are adopting top‐down proteomic workflows. In this review, some recent advances are outlined and current challenges and future directions for the field are discussed.     

Multiplexed Quantitation of Endogenous Proteins in Dried Blood Spots by Multiple Reaction Monitoring - Mass Spectrometry

Dried blood spot (DBS) sampling, coupled with multiple reaction monitoring mass spectrometry (MRM-MS), is a well-established approach for quantifying a wide range of small molecule biomarkers and drugs. This sampling procedure is simpler and less-invasive than those required for traditional plasma or serum samples enabling collection by minimally trained personnel. Many analytes are stable in the DBS format without refrigeration, which reduces the cost and logistical challenges of sample collection in remote locations. These advantages make DBS sample collection desirable for advancing personalized medicine through population-wide biomarker screening. Here we expand this technology by demonstrating the first multiplexed method for the quantitation of endogenous proteins in DBS samples. A panel of 60 abundant proteins in human blood was targeted by monitoring proteotypic tryptic peptides and their stable isotope-labeled analogs by MRM. Linear calibration curves were obtained for 40 of the 65 peptide targets demonstrating multiple proteins can be quantitatively extracted from DBS collection cards. The method was also highly reproducible with a coefficient of variation of <15% for all 40 peptides. Overall, this assay quantified 37 proteins spanning a range of more than four orders of magnitude in concentration within a single 25 min LC/MRM-MS analysis. The protein abundances of the 33 proteins quantified in matching DBS and whole blood samples showed an excellent correlation, with a slope of 0.96 and an R² value of 0.97. Furthermore, the measured concentrations for 80% of the proteins were stable for at least 10 days when stored at −20 °C, 4 °C and 37 °C. This work represents an important first step in evaluating the integration of DBS sampling with highly-multiplexed MRM for quantitation of endogenous proteins.Dried Blood Spot (DBS)¹ samples have many advantages over blood serum or plasma and are the preferred clinical sample for newborn screening for metabolic diseases (1, 2). These samples are collected by pricking a newborn''s heel and spotting a drop of blood onto specially designed filter paper collection cards. Samples are then dried under ambient conditions and are usually stored with desiccant at room temperature until analysis. This sampling procedure is simpler and less invasive then intravenous blood draws, which require a trained phlebotomist. Not surprisingly, the majority of adult patients prefer the small lancet used in finger-prick blood sampling methods to the larger needles used in intravenous blood draws (3, 4). Unlike plasma or serum samples, which consume ≥250 μl of blood and must be centrifuged within an hour of collection, DBS samples can be prepared using a volume of only 10 μl, and do not require any specialized equipment at the collection site (5). The simplicity and reduced safety risks associated with DBS sampling enables collection by minimally trained staff or by the patients themselves. In addition, many analytes are stable in the DBS format at room temperature, reducing sample transportation and storage costs, as well as the impact on the environment. Finally, DBS samples are safer to transport and are considered exempt from dangerous goods regulations (6, 7). These advantages make DBS sampling very attractive for advancing personalized medicine and population-based biomarker research (8).Numerous biomolecular targets covering genomics, metabolomics, and proteomics applications have been quantified in DBS samples using a wide array of analytical techniques (9). The most common clinical application of DBS sampling is screening newborns for metabolomics disorders by targeting small molecule biomarkers. Early screening programs relied on bacterial inhibition assays and later immunoassays, both of which required a different assay for each target of interest (2). However, the time and cost required to perform each assay independently has limited the number of diseases that could be screened nationwide to only a handful. In addition, a single biomarker often lacked the specificity to produce a definitive diagnosis, requiring extensive secondary testing. Hemoglobin is the only protein that is commonly targeted in DBS samples, and primary screening is accomplished by high-performance liquid chromatography (HPLC) or isoelectric focusing (IEF) methods (2). Similar to small-molecule screening methods, these approaches are low-throughput and are not amendable to multiplexing with additional protein targets. In newborn screening programs, these challenges associated with small molecule analysis were overcome with the introduction of multiple reaction monitoring mass spectrometry (MRM-MS) into the clinical laboratories (1, 10). The specificity of MRM enables hundreds of analytes to be monitored during a single experiment to facilitate the development of highly multiplexed assays. The addition of stable isotope-labeled internal standards (SIS) enables the acquisition of highly reproducible results across a variety of instrumentation at different institutions. It is now common for 20–30 small molecule targets including amino acids, fatty acid acylcarnitines, and organic acid acylcarnitines to be analyzed by flow injection MRM-MS, at a cost of $10–20 USD per patient sample (11). Expansion of the screening panel to include additional small-molecule biomarkers on an existing platform may cost less than $1 each. In addition to newborn screening, DBS sampling combined with MS is also gaining acceptance in small-molecule drug development (12, 13). Here the collection of smaller blood volumes allows serial sampling from mice reducing the total number of animals required to generate preclinical toxicology and pharmacokinetic data (5).Despite the successful use of DBS samples in MS-based experiments for small molecule analysis, there have been few reports of using this technology for protein targets (13). Daniel and coworkers reported a screening method for identifying β-thalassemia using the well-known biomarker HbA₂, a hemoglobin variant composed of two alpha- and two delta-globin subunits (14). Proteins were extracted in an aqueous solution, digested with trypsin in 30 min and infused for MRM-MS analysis. Multiple hemoglobin peptides were targeted to measure the abundance of the delta-globin chain, using peptides from the beta-globin chain as an internal standard. This ratio correlated well with the abundance of intact HbA₂ as determined by a well-established HPLC method. The shorter acquisition time and the increased specificity of the MS-based method showed promise for improving population-wide screening. Boemer et al. used a similar flow injection MRM-MS strategy to screen newborns for hemoglobin variants associated with sickle cell disease (15). They analyzed more than 2000 DBS samples by targeting tryptic peptides that were unique to four different beta-globin mutations and compared their results with a standard IEF method that measured the intact proteins obtained from corresponding whole blood samples. Their flow injection MRM approach was able to identify the correct phenotype for all targeted variants. Recently, deWilde et al. reported a method for screening newborn DBS samples for ceruloplasmin, a protein linked to Wilson''s disease (16). Their method combined SIS peptides with LC/MRM-MS and produced results similar to those from an immunoassay for the analysis of seven patient samples. The monitoring of a therapeutic protein in rat blood was demonstrated with Kehler et al. to evaluate the suitability of this approach for supporting preclinical trials (17). Finally, a multiplexed approach was presented by Sleczka et al. for the simultaneous quantitation of two therapeutic proteins in spiked DBS samples collected from several animal models (18).In all previous methods, only one to two proteins were targeted in DBS samples and therefore the true multiplexing capabilities of MRM were not realized. MRM-based methods using SIS peptides have already proven proficient at highly multiplexed quantitation of proteins in plasma and serum samples (19–21). Our current work demonstrates the potential for integrating DBS sampling with LC/MRM-MS for highly multiplexed quantitation of endogenous proteins. Many of the 60 proteins that we have targeted have been cleared or approved by FDA, and are already being analyzed one at a time in clinical laboratories. The assay developed in this study includes a highly reproducible method for extracting multiple proteins from DBS samples followed by trypsin digestion and surrogate peptides, along with their SIS analogs, were analyzed by a standard-flow LC/MRM-MS platform that has previously been shown to give accurate, sensitive, and robust analysis of proteotypic peptides in human plasma (21, 22). The quantitative results from whole blood and the corresponding DBS samples were compared, and the integrity of DBS samples stored under various temperatures has been evaluated.     

Identification of Cell Cycle Dependent Interaction Partners of the Septins by Quantitative Mass Spectrometry

The septins are a conserved family of GTP-binding proteins that, in the baker''s yeast, assemble into a highly ordered array of filaments at the mother bud neck. These filaments undergo significant structural rearrangements during the cell cycle. We aimed at identifying key components that are involved in or regulate the transitions of the septins. By combining cell synchronization and quantitative affinity-purification mass-spectrometry, we performed a screen for specific interaction partners of the septins at three distinct stages of the cell cycle. A total of 83 interaction partners of the septins were assigned. Surprisingly, we detected DNA-interacting/nuclear proteins and proteins involved in ribosome biogenesis and protein synthesis predominantly present in alpha-factor arrested that do not display an assembled septin structure. Furthermore, two distinct sets of regulatory proteins that are specific for cells at S-phase with a stable septin collar or at mitosis with split septin rings were identified.Complementary methods like SPLIFF and immunoprecipitation allowed us to more exactly define the spatial and temporal characteristics of selected hits of the AP-MS screen.     

双向电泳结合质谱分析长双歧杆菌XY01分泌蛋白质图谱

菌体的分泌蛋白质在宿主和菌体的相互作用之间起着重要的作用. 本研究采用双向凝胶电泳的方法建立了长双歧杆菌XY01分泌蛋白质图谱,通过MALDI-TOF/TOF质 谱鉴定和数据库搜索,对鉴定到的分泌蛋白进行了分析. 共检测到21个蛋白质点, 成功鉴定18个蛋白质点,分别代表14个不同的蛋白质,等电点分布在4.5～7.0之间 ,分子质量分布在20 ～65 kD之间;通过COGs分类和功能分析,信号肽和细胞定位及KEGG代谢通路分析. 结果表明,这些蛋白质对菌体细胞壁/膜的形成、生物信号传导和物质代谢等起着重要作用. 研究结果为长双歧杆菌蛋白质组学和基因组学的研究提供了参考.     

Easy Identification of Leishmania Species by Mass Spectrometry

Background

Cutaneous leishmaniasis is caused by several Leishmania species that are associated with variable outcomes before and after therapy. Optimal treatment decision is based on an accurate identification of the infecting species but current methods to type Leishmania isolates are relatively complex and/or slow. Therefore, the initial treatment decision is generally presumptive, the infecting species being suspected on epidemiological and clinical grounds. A simple method to type cultured isolates would facilitate disease management.

Methodology

We analyzed MALDI-TOF spectra of promastigote pellets from 46 strains cultured in monophasic medium, including 20 short-term cultured isolates from French travelers (19 with CL, 1 with VL). As per routine procedure, clinical isolates were analyzed in parallel with Multilocus Sequence Typing (MLST) at the National Reference Center for Leishmania.

Principal Findings

Automatic dendrogram analysis generated a classification of isolates consistent with reference determination of species based on MLST or hsp70 sequencing. A minute analysis of spectra based on a very simple, database-independent analysis of spectra based on the algorithm showed that the mutually exclusive presence of two pairs of peaks discriminated isolates considered by reference methods to belong either to the Viannia or Leishmania subgenus, and that within each subgenus presence or absence of a few peaks allowed discrimination to species complexes level.

Conclusions/Significance

Analysis of cultured Leishmania isolates using mass spectrometry allows a rapid and simple classification to the species complex level consistent with reference methods, a potentially useful method to guide treatment decision in patients with cutaneous leishmaniasis.     

质谱法检测食源性致病菌技术研究进展

食源性致病菌存在广泛,能够引起人类的疾病甚至死亡,研究发现超过一半的食品安全问题来源于食源性致病菌的污染。如何快速有效地检测出食源性致病菌是预防和控制食品安全问题的关键环节。系统地介绍了检测食源性致病菌的方法,包括传统培养法、代谢学法、分子生物学法、免疫学方法等传统方法以及新兴的质谱法。质谱法有检测效率高、操作简便、灵敏度高等优点,着重对质谱法的原理、应用以及未来的发展趋势进行了阐述,以期为该技术的研究开发和推广应用提供参考。     

PILOT_PROTEIN: Identification of Unmodified and Modified Proteins via High-Resolution Mass Spectrometry and Mixed-Integer Linear Optimization

A novel protein identification framework, PILOT_PROTEIN, has been developed to construct a comprehensive list of all unmodified proteins that are present in a living sample. It uses the peptide identification results from the PILOT_SEQUEL algorithm to initially determine all unmodified proteins within the sample. Using a rigorous biclustering approach that groups incorrect peptide sequences with other homologous sequences, the number of false positives reported is minimized. A sequence tag procedure is then incorporated along with the untargeted PTM identification algorithm, PILOT_PTM, to determine a list of all modification types and sites for each protein. The unmodified protein identification algorithm, PILOT_PROTEIN, is compared to the methods SEQUEST, InsPecT, X!Tandem, VEMS, and ProteinProspector using both prepared protein samples and a more complex chromatin digest. The algorithm demonstrates superior protein identification accuracy with a lower false positive rate. All materials are freely available to the scientific community at http://pumpd.princeton.edu .     

Localized Sampling Enables Monitoring of Cell State via Inline Electrospray Ionization Mass Spectrometry

Nascent advanced therapies, including regenerative medicine and cell and gene therapies, rely on the production of cells in bioreactors that are highly heterogeneous in both space and time. Unfortunately, advanced therapies have failed to reach a wide patient population due to unreliable manufacturing processes that result in batch variability and cost prohibitive production. This can be attributed largely to a void in existing process analytical technologies (PATs) capable of characterizing the secreted critical quality attribute (CQA) biomolecules that correlate with the final product quality. The Dynamic Sampling Platform (DSP) is a PAT for cell bioreactor monitoring that can be coupled to a suite of sensor techniques to provide real-time feedback on spatial and temporal CQA content in situ. In this study, DSP is coupled with electrospray ionization mass spectrometry and direct-from-culture sampling to obtain measures of CQA content in bulk media and the cell microenvironment throughout the entire cell culture process (≈3 weeks). Post hoc analysis of this real-time data reveals that sampling from the microenvironment enables cell state monitoring (e.g., confluence, differentiation). These results demonstrate that an effective PAT should incorporate both spatial and temporal resolution to serve as an effective input for feedback control in biomanufacturing.