Yeast Cell Lysate Research Articles

High-Yield Expressed Human Ferritin Heavy-Chain Nanoparticles in K. marxianus for Functional Food Development.

The use of Generally Recognized as Safe (GRAS)-grade microbial cell factories to produce recombinant protein-based nutritional products is a promising trend in developing food and health supplements. In this study, GRAS-grade Kluyveromyces marxianus was employed to express recombinant human heavy-chain ferritin (rhFTH), achieving a yield of 11 g/L in a 5 L fermenter, marking the highest yield reported for ferritin nanoparticle proteins to our knowledge. The rhFTH formed 12 nm spherical nanocages capable of ferroxidase activity, which involves converting Fe2+ to Fe3+ for storage. The rhFTH-containing yeast cell lysates promoted cytokine secretion (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and -1β (IL-1β)) and enhanced locomotion, pharyngeal pumping frequency, egg-laying capacity, and lifespan under heat and oxidative stress in the RAW264.7 mouse cell line and the C. elegans model, respectively, whereas yeast cell lysate alone had no such effects. These findings suggest that rhFTH boosts immunity, holding promise for developing ferritin-based food and nutritional products and suggesting its adjuvant potential for clinical applications of ferritin-based nanomedicine. The high-yield production of ferritin nanoparticles in K. marxianus offers a valuable source of ferritin for the development of ferritin-based products.

A simple and efficient approach for preparing cationic coating with tunable electroosmotic flow for capillary zone electrophoresis-mass spectrometry-based top-down proteomics

BackgroundCapillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has become a valuable analytical technique in top-down proteomics (TDP). CZE-MS/MS-based TDP typically employs separation capillaries with neutral coatings (i.e., linear polyacrylamide, LPA). However, issues related to separation resolution and reproducibility remain with the LPA-coated capillaries due to the unavoidable non-specific protein adsorption onto the capillary wall. Cationic coatings can be critical alternatives to LPA coating for CZE-MS/MS-based TDP due to the electrostatic repulsion between the positively charged capillary inner wall and proteoform molecules in the acidic separation buffer. Unfortunately, there are only very few studies using cationic coating-based CZE-MS/MS for TDP studies. ResultsIn this work, we aimed to develop a simple and efficient approach for preparing separation capillaries with a cationic coating, i.e., poly (acrylamide-co-(3-acrylamidopropyl) trimethylammonium chloride [PAMAPTAC]) for CZE-MS/MS-based TDP. The PAMAPTAC coating-based CZE-MS produced significantly better separation resolution of proteoforms compared to the traditionally used LPA-coated approach. It achieved reproducible separation and measurement of a simple proteoform mixture and a complex proteome sample (i.e., a yeast cell lysate) regarding migration time, proteoform intensity, and the number of proteoform identifications. The PAMAPTAC coating-based CZE-MS enabled the detection of large proteoforms (≥30 kDa) from the yeast cell lysate reproducibly without any size-based prefractionation. Interestingly, the mobility of proteoforms using the PAMAPTAC coating can be predicted accurately using a simple semi-empirical model. SignificanceThe results render the PAMAPTAC coating as a valuable alternative to the LPA coating to advance CZE-MS-based TDP towards high-resolution separation and highly reproducible measurement of proteoforms in complex samples.

Integration of oleaginous yeast-produced lipids into plant-based milk alternatives

In this study, the production of a novel oleaginous yeast-based milk powder alternative is presented. Three formulations, comprising yeast oil, oleaginous yeast cell lysate, and whole cells, were analyzed and compared to a reference formulation, covering the entire process from fermentation to powder analysis. The whole cell formulation exhibited highest encapsulating efficiency and low surface lipid content, indicating improved stability. Moreover, the formulation with cell lysate and whole cells showed larger oil droplet sizes in emulsions and spray-dried particles, and higher viscosity in rheological studies compared to the reference formulation. Electron microscopic images revealed morphological differences in spray-dried particles, emphasizing the impact of yeast oil integration. Concluding, formulations using oleaginous yeast present a complex system with diverse components. While this research pioneers oleaginous yeast integration into milk alternatives, further experiments are essential for a comprehensive understanding of observed effects and unlocking the full potential of these innovative and novel formulations.

Pilot Evaluation of the Long-Term Reproducibility of Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Top-Down Proteomics of a Complex Proteome Sample.

Mass spectrometry (MS)-based top-down proteomics (TDP) has revolutionized biological research by measuring intact proteoforms in cells, tissues, and biofluids. Capillary zone electrophoresis-tandem MS (CZE-MS/MS) is a valuable technique for TDP, offering a high peak capacity and sensitivity for proteoform separation and detection. However, the long-term reproducibility of CZE-MS/MS in TDP remains unstudied, which is a crucial aspect for large-scale studies. This work investigated the long-term qualitative and quantitative reproducibility of CZE-MS/MS for TDP for the first time, focusing on a yeast cell lysate. Over 1000 proteoforms were identified per run across 62 runs using one linear polyacrylamide (LPA)-coated separation capillary, highlighting the robustness of the CZE-MS/MS technique. However, substantial decreases in proteoform intensity and identification were observed after some initial runs due to proteoform adsorption onto the capillary inner wall. To address this issue, we developed an efficient capillary cleanup procedure using diluted ammonium hydroxide, achieving high qualitative and quantitative reproducibility for the yeast sample across at least 23 runs. The data underscore the capability of CZE-MS/MS for large-scale quantitative TDP of complex samples, signaling its readiness for deployment in broad biological applications. The MS RAW files were deposited in ProteomeXchange Consortium with the data set identifier of PXD046651.

Interaction between Gtr2p and ribosomal Rps31p affects the incorporation of Rps31p into ribosomes of Saccharomyces cerevisiae

In yeast, ras-like small G proteins, Gtr1p and Gtr2p, form heterodimers that affect cell division, detect amino acids, and regulate the activity of TORC1, a protein complex that integrates various signals, including those related to nutrient availability, growth factors, and stress signals. To explore novel roles of Gtr2p, yeast two-hybrid screening was performed using gtr2S23Np, an active form of Gtr2p, which identified Rps31p and Rpl12p as Gtr2p-interacting proteins. In the present study, we found that Gtr2p, but not Gtr1p, interacts with Rps31p, a 40S ribosomal subunit, and a component of the ubiquitin fusion protein Ubi3p, which is essential for the initiation and elongation of translation. In yeast cells expressing gtr2Q66Lp, an inactive form of Gtr2p, the interaction between Rps31p and gtr2Q66Lp, as well as the level of exogenous expression of Rps31p, was reduced. However, the level of exogenous expression of Rpl12p was unaffected. Introducing a mutation in ubiquitin target lysine residues to arginine (rps31-K5R) restored the level of exogenously expressed Rps31p and rescued the rapamycin and caffeine sensitivity of gtr2Q66L cells. Sucrose density gradient centrifugation of yeast cell lysate expressing Rps31p and gtr2Q66Lp revealed that exogenously expressed Rps31p was poorly incorporated, whereas rps31-K5Rp was efficiently incorporated, into ribosomes. These results suggest that Gtr2p influences incorporation of Rps31p into ribosomes and contributes to drug resistance through its interaction with Rps31p.

Interrogating the potential of helical aromatic foldamers for protein recognition.

A biotinylated helical aromatic oligoamide foldamer equivalent in size to a 24mer peptide was designed without any prejudice other than to display various polar and hydrophobic side chains at its surface. It was synthesized on solid phase, its P- and M-helical conformers were separated by HPLC on a chiral stationary phase, and the solid state structure of a non-biotinylated analogue was elucidated by X-ray crystallography. Pull-down experiments from a yeast cell lysate using the foldamer as a bait followed by proteomic analysis revealed potential protein binding partners. Three of these proteins were recombinantly expressed. Biolayer interferometry showed submicromolar binding demonstrating the potential of a given foldamer to have affinity for certain proteins in the absence of design considerations. Yet, binding selectivity was low in all three cases since both P- and M-conformers bound to the proteins with similar affinities.

CZE-MS peptide mapping: To desalt or not to desalt?

BackgroundIn “shotgun” approaches involving high-performance liquid chromatography or capillary zone electrophoresis (CZE), matrix removal prior to sample analysis is considered as an indispensable tool. Despite the fact that CZE offers a high tolerance towards salts, most publications reported on the use of desalting. There seems to be no clear consensus on the utilization of desalting in the CZE-MS community, most probably due to the absence of works addressing the comparison of desalted and non-desalted digests. Our aim was to fill this research gap using protein samples of varying complexity in different sample matrices. ResultsFirst, standard protein digests were analyzed to build the knowledge on the effect of sample clean-up by solid-phase extraction (SPE) pipette tips and the possible stacking phenomena induced by different sample matrices. Desalting led to a somewhat altered peptide profile, the procedure affected mostly the hydrophilic peptides (although not to a devastating extent). Nevertheless, desalting samples allowed remarkable stacking efficiency owing to their low-conductivity sample background, enabling a so-called field-amplified sample stacking phenomenon. Non-desalted samples also produced a stacking event, the mechanism of which is based on transient-isotachophoresis due to the presence of high-mobility ions in the digestion buffer itself. Adding either extra ammonium ions or acetonitrile into the non-desalted digests enhanced the stacking efficiency. A complex sample (yeast cell lysate) was also analyzed with the optimal conditions, which yielded similar tendencies. SignificanceBased on these results, we propose that sample clean-up in the bottom-up sample preparation process prior to CZE-MS analysis can be omitted. The preclusion of desalting can even enhance detection sensitivity, separation efficiency or sequence coverage.

Valorization of spent Brewers yeast in the integrated production of the fungal exopolysaccharide (1 → 6)-β-D-glucan (lasiodiplodan) and single-cell protein

Spent Brewers yeast was studied as a low-cost alternative feedstock for sustainably producing a high value-added microbial β-D-glucan (lasiodiplodan). The potential of using the mycelial biomass (single-cell protein, SCP) generated in the production of β-glucan was also evaluated as a strategy of an integrated biorefining. Five cell lysis protocols were studied to obtain a yeast cell lysate rich in nutrients. Mineral supplementation of the fermentation medium was assessed, and the SCP produced was characterized by the nutritional composition, amino acid, and lipid profiles. Lasiodiplodia theobromae MMPI was effective in the co-production of lasiodiplodan ((1 → 6)-β-D-glucan)) and SCP in a cell lyzate-based medium. Enzymatic lysis with Alcalase promoted more significant cell rupture and release of soluble intracellular components. Highest production of lasiodiplodan (2.12 gL-1) and mycelial biomass (9.95 gL-1) resulted from Alcalase-treated cell lysate-based medium (7 gproteinL−1) supplemented with sucrose (60 gL-1). Under these conditions, volumetric productivities of lasiodiplodan and mycelial biomass: 0.029 gL−1h−1 and 0.138 gL−1h−1, respectively, were obtained. Mycelial biomass generated in this cultivation was rich in protein and contained all essential amino acids, especially high concentrations of leucine (69 mgg−1protein), lysine (62.2 mgg−1protein), valine (44.2 mgg−1protein), threonine (43.2 mgg−1protein). Co-production of lasiodiplodan and SCP could be an economically promising bioprospecting alternative.

Pilot investigation of magnetic nanoparticle-based immobilized metal affinity chromatography for efficient enrichment of phosphoproteoforms for mass spectrometry-based top-down proteomics.

Protein phosphorylation is a vital and common post-translational modification (PTM) in cells, modulating various biological processes and diseases. Comprehensive top-down proteomics of phosphorylated proteoforms (phosphoproteoforms) in cells and tissues is essential for a better understanding of the roles of protein phosphorylation in fundamental biological processes and diseases. Mass spectrometry (MS)-based top-down proteomics of phosphoproteoforms remains challenging due to their relatively low abundance. Herein, we investigated magnetic nanoparticle-based immobilized metal affinity chromatography (IMAC, Ti4+, and Fe3+) for selective enrichment of phosphoproteoforms for MS-based top-down proteomics. The IMAC method achieved reproducible and highly efficient enrichment of phosphoproteoforms from simple and complex protein mixtures. It outperformed one commercial phosphoprotein enrichment kit regarding the capture efficiency and recovery of phosphoproteins. Reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) analyses of yeast cell lysates after IMAC (Ti4+ or Fe3+) enrichment produced roughly 100% more phosphoproteoform identifications compared to without IMAC enrichment. Importantly, the phosphoproteoforms identified after Ti4+-IMAC or Fe3+-IMAC enrichment correspond to proteins with much lower overall abundance compared to that identified without the IMAC treatment. We also revealed that Ti4+-IMAC and Fe3+-IMAC could enrich different pools of phosphoproteoforms from complex proteomes and the combination of those two methods will be useful for further improving the phosphoproteoform coverage from complex samples. The results clearly demonstrate the value of our magnetic nanoparticle-based Ti4+-IMAC and Fe3+-IMAC for advancing top-down MS characterization of phosphoproteoforms in complex biological systems.

Bottom-Up Synthetic Biology Using Cell-Free Protein Synthesis.

Technical advances in biotechnology have greatly accelerated the development of bottom-up synthetic biology. Unlike top-down approaches, bottom-up synthetic biology focuses on the construction of a minimal cell from scratch and the application of these principles to solve challenges. Cell-free protein synthesis (CFPS) systems provide minimal machinery for transcription and translation, from either a fractionated cell lysate or individual purified protein elements, thus speeding up the development of synthetic cell projects. In this review, we trace the history of the cell-free technique back to the first in vitro fermentation experiment using yeast cell lysate. Furthermore, we summarized progresses of individual cell mimicry modules, such as compartmentalization, gene expression regulation, energy regeneration and metabolism, growth and division, communication, and motility. Finally, current challenges and future perspectives on the field are outlined.

Iron Mobilization from Ferritin in Yeast Cell Lysate and Physiological Implications.

Most in vitro iron mobilization studies from ferritin have been performed in aqueous buffered solutions using a variety of reducing substances. The kinetics of iron mobilization from ferritin in a medium that resembles the complex milieu of cells could dramatically differ from those in aqueous solutions, and to our knowledge, no such studies have been performed. Here, we have studied the kinetics of iron release from ferritin in fresh yeast cell lysates and examined the effect of cellular metabolites on this process. Our results show that iron release from ferritin in buffer is extremely slow compared to cell lysate under identical experimental conditions, suggesting that certain cellular metabolites present in yeast cell lysate facilitate the reductive release of ferric iron from the ferritin core. Using filtration membranes with different molecular weight cut-offs (3, 10, 30, 50, and 100 kDa), we demonstrate that a cellular component >50 kDa is implicated in the reductive release of iron. When the cell lysate was washed three times with buffer, or when NADPH was omitted from the solution, a dramatic decrease in iron mobilization rates was observed. The addition of physiological concentrations of free flavins, such as FMN, FAD, and riboflavin showed about a two-fold increase in the amount of released iron. Notably, all iron release kinetics occurred while the solution oxygen level was still high. Altogether, our results indicate that in addition to ferritin proteolysis, there exists an auxiliary iron reductive mechanism that involves long-range electron transfer reactions facilitated by the ferritin shell. The physiological implications of such iron reductive mechanisms are discussed.

Clionamines stimulate autophagy, inhibit Mycobacterium tuberculosis survival in macrophages, and target Pik1

The pathogen Mycobacterium tuberculosis (Mtb) evades the innate immune system by interfering with autophagy and phagosomal maturation in macrophages, and, as a result, small molecule stimulation of autophagy represents a host-directed therapeutics (HDTs) approach for treatment of tuberculosis (TB). Here we show the marine natural product clionamines activate autophagy and inhibit Mtb survival in macrophages. A yeast chemical-genetics approach identified Pik1 as target protein of the clionamines. Biotinylated clionamine B pulled down Pik1 from yeast cell lysates and a clionamine analog inhibited phosphatidyl 4-phosphate (PI4P) production in yeast Golgi membranes. Chemical-genetic profiles of clionamines and cationic amphiphilic drugs (CADs) are closely related, linking the clionamine mode of action to co-localization with PI4P in a vesicular compartment. Small interfering RNA (siRNA) knockdown of PI4KB, a human homolog of Pik1, inhibited the survival of Mtb in macrophages, identifying PI4KB as an unexploited molecular target for efforts to develop HDT drugs for treatment of TB.

MsmsEDA & msmsTests: Label-Free Differential Expression by Spectral Counts.

msmsTests is an R/Bioconductor package providing functions for statistical tests in label-free LC-MS/MS data by spectral counts. These functions aim at discovering differentially expressed proteins between two biological conditions. Three tests are available: Poisson GLM regression, quasi-likelihood GLM regression, and the negative binomial of the edgeR package. The three models admit blocking factors to control for nuisance variables. To assure a good level of reproducibility a post-test filter is available, where (1) a minimum effect size considered biologically relevant, and (2) a minimum expression of the most abundant condition, may be set. A companion package, msmsEDA, proposes functions to explore datasets based on msms spectral counts. The provided graphics help in identifying outliers, the presence of eventual batch factors, and check the effects of different normalizing strategies. This protocol illustrates the use of both packages on two examples: A purely spike-in experiment of 48 human proteins in a standard yeast cell lysate; and a cancer cell-line secretome dataset requiring a biological normalization.

A simple method to purify recombinant HCV core protein expressed in Pichia pastoris for obtaining virus-like particles and producing monoclonal antibodies

In this study, we describe an optimized method of obtaining virus-like particles (VLPs) of the recombinant hepatitis C virus (HCV) core protein (HCcAg) expressed in yeast cells (Pichia pastoris), which can be used for the construction of diagnostic test systems and vaccine engineering. The described simplified procedure was developed to enable in vitro self-assembly of HCcAg molecules into VLPs during protein purification. In brief, the HCcAg protein was precipitated from yeast cell lysates with ammonium sulfate and renatured by gel filtration on Sephadex G-25 under reducing conditions. VLPs were self-assembled after the removal of the reducing agent by gel filtration on Sephadex G-25. Protein purity and specificity were evaluated by SDS-PAGE and immunoblotting analysis. The molecular mass of VLPs and their relative quantity were measured by HPLC, followed by confirmation of VLPs production and estimation of their shape and size by transmission electron microscopy. As a result, we obtained recombinant HCcAg preparation (with ~90% purity) in the form of VLPs and monomers, which has been used to produce hybridomas secreting monoclonal antibodies (mAbs) against HCcAg.

Characterization of β-Galactosidase from Lactose Utilizing Yeast Isolated from the Dairy Sample

Objectives: The objective of the study was to isolate lactose positive yeasts from dairy samples collected from local markets of Kathmandu, to extract crude β-galactosidase from the lactose positive yeast and to characterize the enzyme for optimum time duration, pH, temperature, Michaelis-Menten constant (Km) and maximum activity (Vmax).
 Methods: Four lactose positive yeast strains were isolated from dairy samples collected from local market of Kathmandu by pour plate method. Single strain having maximum lactose positive activity was selected for the study. The mass culture of the lactose positive yeast strain was lysed by 2% Chloroform and the yeast cell lysate containing β-galactosidase (i.e. crude enzyme extract) was characterized by using ONPG (Ortho-Nitrophenyl-β-D-galactopyranoside) as substrate. ONPG is a colorless substrate for the enzyme assay which is hydrolyzed by the enzyme into yellow colored product ONP (Ortho-Nitrophenol). The concentration of product formed was monitored spectrophotometrically at 420 nm to determine the enzyme activity and to characterize the enzyme.
 Results: The enzyme had wide range of working temperature from 0-50ºC, with optimal temperature of 37ºC. However, greater than 50% hydrolyzing ability was maintained in the range of 14-40ºC. Optimum time of reaction was 70 min. The enzyme had maximum activity in the near neutral pH of 6.8. Michaelis-Menten constant of the enzyme was found to be 2.23 mM of ONPG and Vmax was 58.82 nmol/min/ml. Enzyme activity was 27.88 nmol/min/ml, Specific enzyme activity was 59.97 nmol/ min/mg and total enzyme activity was 3346.33 nmol/min.
 Conclusion: The activity over a wide range of temperature 0-50ºC with low Km value shows that the enzyme has a commercial application in clearance of lactose pollution in waste water in different environmental conditions.

Formation of glutathione patulin conjugates associated with yeast fermentation contributes to patulin reduction

Patulin (PAT) is a fungal secondary metabolite which is mainly detected in rotten apples, and possesses serious risks to human health. Saccharomyces cerevisiae has become a promising biological control candidate due to its ability to degrade PAT. However, the degradation mechanism remains elusive. In this study, the degradation system and potential other metabolisms of PAT by S. cerevisiae were investigated. After a 96 h fermentation, 5.0 mg/L of PAT was completely metabolized by S. cerevisiae. Among the mixture of metabolites, ascladiol was identified but only accounted for 3.3% of the initial PAT. Yeast cell lysates, attained from a 96 h culture, altered 10 mg/L of PAT within 8 h. As this activity was reduced by heat treatment, it indicated that soluble intracellular enzymes were key components in PAT transformation. In addition, these effective intracellular enzymes were obtained in PAT-treated yeast cells instead of the untreated cells, indicating the activity was induced by PAT. LC-MS/MS revealed a drastic decrease of glutathione (GSH) in cell lysate upon addition of PAT. Several glutathione-patulin (GSH-PAT) conjugates (mainly c-GSH-PAT and l-GSH-PAT) were later identified. The conjugation of GSH and PAT plays an important role in PAT reduction by yeast cell lysate, even after enzymes in the lysate have been heat inactivated. Therefore, the PAT reduction by S. cerevisiae was a consequence of PAT-induced intracellular enzymes and GSH-mediated conjugation.

DNA purification and concentration by isotachophoresis in nonwoven fabric strip

We present a novel method for concentration and purification of DNA from biological samples. The method is based on isotachophoretic separation of DNA strands in a separation bed made of a disposable nonwoven fabric strip. Application of oxalate as the leading ion prevented corrosion of the carbon anode and also the leading ion was continually removed from the system due to its decomposition into CO2 at the anode. The fractions were marked by three colored markers of electrophoretic mobility closely surrounding the mobility of DNA. The fraction collection was realized by a centrifugal drain of cut out strip segments. The method was evaluated using two purified salmon sperm DNA fragments of lengths 200 bp and 2000 bp. The results confirmed the high DNA concentrating effect of the method (34-fold increase of the original DNA concentration). The composition of running solutions and voltage program were optimized in order to finish the analysis within 30 min. The optimized method was used to extract, concentrate and purify DNA from a crude yeast cell lysate. The maximum DNA enrichment factor decreased to 12 due to the stretching of DNA zones caused by low-molecular contaminants present in the original lysate. The average recovery determined for yeast DNA was 71 ± 11% (n = 3). The connected elimination of the proteins from DNA zones resulted in the purification factor value of 582 for DNA vs proteins. This demonstrates that the presented method is capable to concentrate DNA from the bulk volume and to further purify it from crude cell lysates using a simple instrumentation and low-cost disposable separation bed.

Chemo-Selection Strategy for Limited Proteolysis Experiments on the Proteomic Scale

Described here is a chemo-selective enrichment strategy, termed the semitryptic peptide enrichment strategy for proteolysis procedures (STEPP), to isolate the semitryptic peptides generated in mass spectrometry-based proteome-wide applications of limited proteolysis methods. The strategy involves reacting the ε-amino groups of lysine side chains and any N-termini created in the limited proteolysis reaction with isobaric mass tags. A subsequent digestion of the sample with trypsin and the chemo-selective reaction of the newly exposed N-termini of the tryptic peptides with N-hydroxysuccinimide (NHS)-activated agarose resin removes the tryptic peptides from solution, leaving only the semitryptic peptides with one nontryptic cleavage site generated in the limited proteolysis reaction for subsequent LC-MS/MS analysis. As part of this work, the STEPP technique is interfaced with two different proteolysis methods, including the pulse proteolysis (PP) and limited proteolysis (LiP) methods. The STEPP-PP workflow is evaluated in two proof-of-principle experiments involving the proteins in a yeast cell lysate and two well-studied drugs, cyclosporin A and geldanamycin. The STEPP-LiP workflow is evaluated in a proof-of-principle experiment involving the proteins in two cell culture models of human breast cancer, MCF-7 and MCF-10A cell lines. The STEPP protocol increased the number of semitryptic peptides detected in the LiP and PP experiments by 5- to 10-fold. The STEPP protocol not only increases the proteomic coverage, but also increases the amount of structural information that can be gleaned from limited proteolysis experiments. Moreover, the protocol also enables the quantitative determination of ligand binding affinities.

Identification and Quantification of Murine Mitochondrial Proteoforms Using an Integrated Top-Down and Intact-Mass Strategy.

The development of effective strategies for the comprehensive identification and quantification of proteoforms in complex systems is a critical challenge in proteomics. Proteoforms, the specific molecular forms in which proteins are present in biological systems, are the key effectors of biological function. Thus, knowledge of proteoform identities and abundances is essential to unraveling the mechanisms that underlie protein function. We recently reported a strategy that integrates conventional top-down mass spectrometry with intact-mass determinations for enhanced proteoform identifications and the elucidation of proteoform families and applied it to the analysis of yeast cell lysate. In the present work, we extend this strategy to enable quantification of proteoforms, and we examine changes in the abundance of murine mitochondrial proteoforms upon differentiation of mouse myoblasts to myotubes. The integrated top-down and intact-mass strategy provided an increase of ∼37% in the number of identified proteoforms compared to top-down alone, which is in agreement with our previous work in yeast; 1779 unique proteoforms were identified using the integrated strategy compared to 1301 using top-down analysis alone. Quantitative comparison of proteoform differences between the myoblast and myotube cell types showed 129 observed proteoforms exhibiting statistically significant abundance changes (fold change >2 and false discovery rate <5%).

Enrichment strategy of protein N-termini based on dioctyl labeling

The analysis of protein N-termini provides valuable information for protein structure and function annotation, and helps the profiling of proteases substrates and cleavage sites. However, most of the current N-terminal enrichment approaches involve multiple chromatographic separation processes and require a large amount of scavenger materials, which make it time and labor consuming and may induce significant sample loss. Herein, we develop a negative N-termini enrichment strategy based on dioctyl labeling. With yeast lysate digests as the sample, the strategy showed a high efficiency in dioctyl labeling and retention shift. Such a strategy was applied for the enrichment of N-terminal peptides from yeast cell lysates and enabled the identification of 237 original N-termini and 133 neo-N-termini, which was improved by 100% and 480% compared with direct analysis. Furthermore, with the combination of multi-proteases digestion, the number of protein N-termini and neo-N-termin was improved by 50% and 90%, respectively, facilitating the in-depth N-terminome analysis.