Direct Identification of Intact Proteins Using a Low-Resolution Mass Spectrometer with CIDn/ETnoD.

Over the past decades, proteomics has become increasingly important and a heavily discussed topic. The identification of intact proteins remains a major focus in this field. While most intact proteins are analyzed using high-resolution mass spectrometry, identifying them through low-resolution mass spectrometry continues to pose challenges. In our study, we investigated the capability of identifying various intact proteins using collision-induced dissociation (CID) and electron transfer without dissociation (ETnoD). Using myoglobin as our test protein, stable product ions were generated with CID, and the identities of the product ions were identified with ETnoD. ETnoD uses a short activation time (AcT, 5 ms) to create sequential charge-reduced precursor ion (CRI). The charges of the fragments and their sequences were determined with corresponding CRI. The product ions can be selected for subsequent CID (termed CIDn) combined with ETnoD for further sequence identification and validation. We refer to this method as CIDn/ETnoD. The use of a multistage CID activation (CIDn) and ETnoD protocol has been applied to several intact proteins to obtain multiple sequence identifications.

Electrochemical and Plasmonic Detection of Myocardial Infarction Using Microfluidic Biochip Incorporated with Mesoporous Nanoscaffolds.

This paper reports a microfluidic device for the electrochemical and plasmonic detection of cardiac myoglobin (cMb) and cardiac troponin I (cTnI) with noticeable limits of detection (LoD) as low as a few picograms per milliliter (pg/mL) ranges, achieved in a short detection time. The device features two working electrodes, each with a mesoporous Ni3V2O8 nanoscaffold grafted with reduced graphene oxide (rGO) that improves the interaction of diffusing analyte molecules with the sensing surface by providing a high surface area and reaction kinetics. Electrochemical studies reveal sensitivities as high as 9.68 µA ng/mL and a LoD of 2.0 pg/mL for cTnI, and 8.98 µA ng/mL and 4.7 pg/mL for cMb. Additionally, the surface plasmon resonance (SPR) studies demonstrate a low-level LoD of 8.8 pg/mL for cMb and 7.3 pg/mL for cTnI. The dual-modality sensor enables dynamic tracking of kinetic antigen-antibody interactions during sensing, self-verification through providing signals of two modes, and reduced false readout. This study demonstrates the complementary nature of the electrochemical and SPR modes in biosensing, with the electrochemical mode being highly sensitive and the SPR mode providing superior tracking of molecular recognition behaviors. The presented sensor represents a significant innovation in cardiovascular disease management and can be applied to monitor other clinically important biomolecules.

Electrochemical detection of myoglobin using an ultrasensitive label-free sensor derived from cubic-ZIF67@Au-rGOF-NH2 composite of MOF and GOF.

Markers of myocardial injury, such as myoglobin (Mb), are substances swiftly released into the peripheral bloodstream upon myocardial cell injury or altered cardiac activity. During the onset of acute myocardial infarction, patients experience a significant surge in serum Mb levels. Given this, precise detection of Mb is essential, necessitating the development of innovative assays to optimize detection capabilities. This study introduces the synthesis of a three-dimensional hierarchical nanocomposite, Cubic-ZIF67@Au-rGOF-NH2, utilizing aminated reduced graphene oxide and zeolite imidazolium ester framework-67 (ZIF67) as foundational structures. Notably, this novel material, applied in a label-free electrochemical immunosensor, presents a groundbreaking approach for detecting myocardial injury markers. Experimental outcomes revealed ZIF67 and AuNPs exhibit enhanced affinity and growth on the 3D-rGOF-NH2 matrix, thus amplifying electrical conductivity while preserving the inherent electrochemical attributes of ZIF67. As a result, the Cubic-ZIF67@Au-rGOF-NH2 label-free electrochemical immunosensor exhibited a broad detection range and high sensitivity for Mb. The derived standard curve was ΔIp = 16.67552lgC+275.245 (R = 0.993) with a detection threshold of 3.47 fg/ml. Moreover, recoveries of standards spiked into samples ranged between 96.3% and 108.7%. Importantly, the devised immunosensor retained notable selectivity against non-target proteins, proving its potential clinical utility based on exemplary sample analysis performance.

Novel needle-probe single-fiber reflectance spectroscopy to quantify sub-surface myoglobin forms in beef psoas major steaks during retail display.

Meat discoloration starts at the interface between the bright red oxymyoglobin layer and the interior deoxymyoglobin layer. Currently, limited tools are available to characterize myoglobin forms formed within the sub-surface of meat. The objective was to demonstrate a needle-probe based single-fiber reflectance (SfR) spectroscopy approach for characterizing sub-surface myoglobin forms of beef psoas major muscles during retail storage. A 400-µm fiber was placed in a 17-gauge needle, and the assembly was inserted into the muscle at five depths of 1 mm increment and 1 cm lateral shift. Metmyoglobin content increased at all depths during display and content at 1 mm was greater compared to that of 2 to 5 mm depth. The a* values decreased (P < 0.05) during retail display aligning with the sub-surface formation of metmyoglobin. In summary, the results suggest that needle-probe SfR spectroscopy can determine interior myoglobin forms and characterize meat discoloration.

Muscle of dark and normal beef differs metabolically.

Reduction in muscle glycogen triggered by adverse antemortem handling events alters postmortem energy metabolism and results in a high ultimate pH and dark, firm and dry beef, often referred to as 'dark-cutting'. However, the relationship between atypical dark (AT) beef, postmortem energy metabolism and underlying tissue characteristics remains somewhat unclear. Cattle harvested in the US and Canada representing normal (pH < 5.6), AT dark (pH 5.6-5.8) and dark cutting (DC; pH > 5.8) beef were analyzed for tissue characteristics related to energy metabolism. Results show AT dark beef is more oxidative but similar to normal beef in glycolytic potential and nucleotide abundance. Mitochondria DNA content (P < 0.05, Canada; P < 0.005, US) and oxidative enzymes for DC and AT dark beef were greater (P < 0.01; Canada and US) compared to normal beef. Myoglobin tracked (P < 0.01) with color classification. These findings show both DC and AT beef are inherently more oxidative and raise the possibility that more oxidative muscle may be more prone to develop dark beef.

Proteomic approaches to characterize biochemistry of fresh beef color.

Color of retail fresh beef is the most important quality influencing the consumers' purchase decisions at the point of sale. Discolored fresh beef cuts are either discarded or converted to low-value products, before the microbial quality is compromised, resulting in huge economic loss to meat industry. The interinfluential interactions between myoglobin, small biomolecules, proteome, and cellular components in postmortem skeletal muscles govern the color stability of fresh beef. This review examines the novel applications of high-throughput tools in mass spectrometry and proteomics to elucidate the fundamental basis of these interactions and to explain the underpinning mechanisms of fresh beef color. Advanced proteomic research indicates that a multitude of factors endogenous to skeletal muscles critically influence the biochemistry of myoglobin and color stability in fresh beef. Additionally, this review highlights the potential of muscle proteome components and myoglobin modifications as novel biomarkers for fresh beef color. SIGNIFICANCE: This review highlights the important role of muscle proteome in fresh beef color, which is the major trait impacting consumers' purchase decisions. In recent years, innovative approaches in proteomics have been exploited for an in-depth understanding of the biochemical mechanisms influencing color development and color stability in fresh beef. The review suggests that a wide range of factors, including endogenous skeletal muscle components, can affect myoglobin biochemistry and color stability in beef. Furthermore, the potential use of muscle proteome components and myoglobin post-translational modifications as biomarkers for fresh beef color is discussed. The currently available body of evidence presented in this review can have important implications in meat industry as it provides novel insights into the factors influencing fresh beef color and an up-to-date list of biomarkers that can be used to predict beef color quality.

A Case of Kidney Transplantation from a Deceased Donor with Acute Kidney Injury due to Rhabdomyolysis.

Acute kidney injury (AKI) due to rhabdomyolysis occurs because of renal ischemia or acute tubular necrosis due to the deposition of myoglobin casts in the renal tubules. Donors with AKI due to rhabdomyolysis are not contraindication for transplantation. However, the dark red kidney raises concerns about renal hypofunction or primary nonfunction after transplantation. We report the case of a 34-year-old man with a 15-year history of hemodialysis for chronic renal failure due to congenital anomalies of the kidney and urinary tract. The patient received a renal allograft from a young woman who suffered cardiac death. The serum creatinine (sCre) level of the donor at the time of transport was 0.6 mg/dL, and renal ultrasonography revealed no abnormalities in renal morphology or blood flow. Her serum creatinine kinase level increased to 57,000 IU/L 58 h after femoral artery cannulation and sCre level worsened to 1.4 mg/dL, suggesting AKI due to rhabdomyolysis. However, since the urine output of the donor was maintained, the sCre elevation was thought to be nonproblematic. The allograft had a dark red appearance at the time of procurement. The perfusion of the isolated kidney was good, but the dark red color did not improve. A 0-h biopsy showed flattening of the renal tubular epithelium and absence of the brush border and myoglobin casts in 30% of the renal tubules. Rhabdomyolysis-related tubular damage was diagnosed. Hemodialysis was discontinued on postoperative day 14. Twenty-four days after the operation, the transplanted kidney function progressed favorably (sCre 1.18 mg/dL), and the patient was discharged. Protocol biopsy 1 month after transplantation showed disappearance of myoglobin casts and improvement in renal tubular epithelial damage. The patient's sCre level was approximately 1.0 mg/dL 24 months after transplantation, and he is doing well without complications.

Kinetics analysis of the reactions responsible for myoglobin chemical state in meat using an advanced reaction-diffusion model.

Here we developed an advanced reaction-diffusion model to predict the evolution of the myoglobin state in beef meat using numerous reactions with rate constants of different orders of magnitude. The initial scheme included 44 reactions from the literature. Sensitivity analysis proved that this initial scheme was equivalent to a simple 22-reaction scheme. Results calculated with this scheme were compared against the spatial distributions of oxymyoglobin (MbO2), metmyoglobin (MMb) and deoxymyoglobin (DMb) measured in meat cuts stored at 20°C under air-permeable packaging. We found global agreement between measured and calculated distributions when adequate rate constant values were used, particularly for the formation of MbO2 from DMb. The model was used to calculate evolutions in MbO2 and MMb distributions under different situations (modified-atmosphere packaging, Fenton chemistry with or without water-soluble antioxidants, increased mitochondrial oxygen consumption). Results were used to discuss the underlying kinetics reaction mechanisms and the performances and limits of the model.

Nanopore-Based Protein Identification.

The implementation of a reliable, rapid, inexpensive, and simple method for whole-proteome identification would greatly benefit cell biology research and clinical medicine. Proteins are currently identified by cleaving them with proteases, detecting the polypeptide fragments with mass spectrometry, and mapping the latter to sequences in genomic/proteomic databases. Here, we demonstrate that the polypeptide fragments can instead be detected and classified at the single-molecule limit using a nanometer-scale pore formed by the protein aerolysin. Specifically, three different water-soluble proteins treated with the same protease, trypsin, produce different polypeptide fragments defined by the degree by which the latter reduce the nanopore's ionic current. The fragments identified with the aerolysin nanopore are consistent with the predicted fragments that trypsin could produce.

Machine Learning Assisted Simultaneous Structural Profiling of Differently Charged Proteins in a Mycobacterium smegmatis Porin A (MspA) Electroosmotic Trap.

The nanopore is emerging as a means of single-molecule protein sensing. However, proteins demonstrate different charge properties, which complicates the design of a sensor that can achieve simultaneous sensing of differently charged proteins. In this work, we introduce an asymmetric electrolyte buffer combined with the Mycobacterium smegmatis porin A (MspA) nanopore to form an electroosmotic flow (EOF) trap. Apo- and holo-myoglobin, which differ in only a single heme, can be fully distinguished by this method. Direct discrimination of lysozyme, apo/holo-myoglobin, and the ACTR/NCBD protein complex, which are basic, neutral, and acidic proteins, respectively, was simultaneously achieved by the MspA EOF trap. To automate event classification, multiple event features were extracted to build a machine learning model, with which a 99.9% accuracy is achieved. The demonstrated method was also applied to identify single molecules of α-lactalbumin and ß-lactoglobulin directly from whey protein powder. This protein-sensing strategy is useful in direct recognition of a protein from a mixture, suggesting its prospective use in rapid and sensitive detection of biomarkers or real-time protein structural analysis.

Vascular rinsing and chilling effects on meat quality attributes from cull dairy cows associated with the two lowest-valued marketing classes.

Commercially harvested cull dairy cow carcasses (n = 64) from the two lowest-valued marketing classes (MC: Lean, LE; Light, LI) were conventionally chilled (CN) or vascularly rinsed with a chilled isotonic substrate solution (Rinse & Chill®; RC). Longissimus lumborum (LL) and Triceps brachii (TB) muscles were processed (steaks, ground). Early postmortem (first 24 h), RC resulted in a lower pH at each time measured. RC steaks had longer sarcomeres and lower shear force than CN. RC produced greater redness associated with blooming and display times. RC LE beef resulted in greater oxymyoglobin during display times. RC ground TB had greater moisture fat-free than CN. RC Lean LL had less purge loss compared to CN LE. RC had greater total pigments than CN. RC ground TB had greater oxygen consumption and lower thiobarbituric acid reactive substances compared to CN. RC has the potential to improve tenderness and color as well as limit lipid oxidation with similar benefits across the two marketing classes.

Evaluating the failure to bloom in dark-cutting and lactate-enhanced beef longissimus steaks.

Previous studies have noted lower L* (lightness) values for both dark-cutting beef and normal-pH beef enhanced with lactate. In the current study, absorption-coefficient, scattering-coefficient, CIE L*a*b* values, refractive index of sarcoplasm, and inter-muscle bundle space were evaluated for dark-cutting beef, normal-pH beef enhanced with lactate, normal-pH beef enhanced with water, and normal-pH beef not enhanced with either water or lactate. Compared with non-enhanced loins, lactate-enhancement had lower a*, chroma, oxymyoglobin, reflectance, scattering, and inter-muscle bundle space as well as greater absorption and refractive index. Dark-cutting steaks had lower a*, chroma, oxymyoglobin values, reflectance, and scattering as well as less inter-muscle bundle space compared with lactate-enhanced steaks. Sarcoplasm refractive index values were greater in lactate-enhanced steaks than dark-cutting steaks. The results suggest that changes in muscle structure and optical properties due to either pH or lactate addition can alter muscle darkening and blooming properties.

Label-Free Electrochemical Sensor Based on Manganese Doped Titanium Dioxide Nanoparticles for Myoglobin Detection: Biomarker for Acute Myocardial Infarction.

A label free electrochemical sensor based on pure titanium oxide and manganese (Mn)-doped titanium oxide (TiO2) nanoparticles are fabricated and characterized for the sensitive detection of myoglobin (Mb) levels to analyze the cardiovascular infarction. Pristine and Mn-doped TiO2 nanoparticles were synthesized via the sol-gel method and characterized in order to understand their structure, morphologies, composition and optical properties. The structural properties revealed that the pure- and doped-TiO2 nanoparticles possess different TiO2 planes. FTIR studies confirm the formation of metal oxide nanoparticles by exhibiting a well-defined peak in the range of 600-650 cm-1. The values of the optical band gap, estimated from UV-Vis spectroscopy, are decreased for the Mn-doped TiO2 nanoparticles. UV-Vis spectra in the presence of myoglobin (Mb) indicated interaction between the TiO2 nanoparticles and myoglobin. The SPE electrodes were then fabricated by printing powder film over the working electrode and tested for label-free electrochemical detection of myoglobin (Mb) in the concentration range of 0-15 nM Mb. The fabricated electrochemical sensor exhibited a high sensitivity of 100.40 µA-cm-2/nM with a lowest detection limit of 0.013 nM (0.22 ng/mL) and a response time of ≤10 ms for sample S3. An interference study with cyt-c and Human Serum Albumin (HSA) of the sensors show the selective response towards Mb in 1:1 mixture.

Reduced Scattering Coefficient During Incremental Exercise Is Constant Without Being Affected by Changes in Muscle Oxygenation or Hemodynamics.

Previous studies have reported that the reduced scattering coefficient (µs') in the vastus lateralis changes during ramp-incremental exercise due to blood volume changes or accumulation of metabolic by-products. We aimed to clarify the influences of deoxygenation and blood volume changes during exercise on µs' dynamics in subjects with various aerobic capacities. Twenty-three healthy young men participated in this study. All subjects performed a ramp-incremental cycling exercise until exhaustion and were divided into two groups: lower (Low: n = 12; peak pulmonary oxygen uptake per kg of fat-free mass (VO2peak), 54.2 ± 5.3 mL/kg/min) and higher aerobic capacity group (High: n = 11; VO2peak, 69.7 ± 5.2 mL/kg/min) by median of VO2peak. Deoxygenated hemoglobin and myoglobin concentrations (deoxy[Hb + Mb]) and total [Hb + Mb] (total[Hb + Mb]) in the vastus lateralis were monitored during the exercise by three-wavelength (760, 800, and 830 nm) time-resolved NIRS. Similarly, µs' at each wavelength was continuously monitored. With increasing exercise intensity, deoxy[Hb + Mb] and total[Hb + Mb] significantly increased in both groups, and the average values of the peak amplitudes of deoxy[Hb + Mb] and total[Hb + Mb] during exercise showed a 106.4% increase and a 17.9% increase from the start of the exercise, respectively. Furthermore, the peak amplitude of total[Hb + Mb] was significantly greater in High. Conversely, there were no changes in µs' at any wavelength during exercise and no differences between two groups, suggesting that the great deoxygenation and blood volume changes during incremental exercise have little effect on µs' dynamics.

Effect of Blood Flow on Hemoglobin and Myoglobin Oxygenation in Contracting Muscle Using Near-Infrared Spectroscopy.

Insufficient O2 delivery to, and uptake by skeletal muscle can produce mobility limitations for patients with chronic diseases. Near-infrared spectroscopy (NIRS) can be used to noninvasively quantify the balance between skeletal muscle O2 delivery and utilization during contraction. However, it is not clear how the oxygenated or deoxygenated NIRS signal should be used to assess muscle O2 changes. This issue is related to the fact that the contributions of hemoglobin (Hb) and myoglobin (Mb) cannot be distinguished. This conundrum can be resolved by quantitative analysis of experimental data by computer simulations with a mechanistic, mathematical model. Model simulations distinguish dynamic responses of the oxygenated (HbO2, MbO2) and deoxygenated (HHb, HMb) contributions to the NIRS signal components (HbMbO2, HHbMb). Simulations of muscle O2 uptake and NIRS kinetics correspond closely to published experimental data (Hernández et al., J Appl Physiol 108: 1169-1176, 2010). Simulated muscle O2 uptake and oxygenation kinetics with different blood flows indicate (1) faster O2 delivery is responsible for slower muscle oxygenation kinetics; (2) Hb and Mb contributions to the HbMbO2 are similar (40-60%); and (3) Hb and Mb contributions to the HHbMb are significantly different, 80% and 20%, respectively. The effect of slow blood flow kinetics on oxygenated Hb and Mb contributions is minimal. However, the effect on the imbalance between O2 delivery and utilization rates causes significant overshoots and undershoots of deoxygenated Hb and Mb contributions. Model analysis in combination with NIRS measurements and information on hemodynamic and microvascular distribution can help to determine the use of NIRS signal in evaluating the factors limiting exercise tolerance in health and disease states.

Rapid and non-destructive spectroscopic method for classifying beef freshness using a deep spectral network fused with myoglobin information.

A simple, novel, rapid, and non-destructive spectroscopic method that employs the deep spectral network for beef-freshness classification was developed. The deep-learning-based model classified beef freshness by learning myoglobin information and reflectance spectra over different freshness states. The reflectance spectra (480-920 nm) were measured from 78 beef samples for 17 days, and the datasets were sorted into three freshness classes based on their pH values. Myoglobin information showed statistically significant differences depending on the freshness; consequently, it was utilized as a crucial parameter for classification. The model exhibited improved performance when the reflectance spectra were combined with the myoglobin information. The accuracy of the proposed model improved to 91.9%, whereas that of the single-spectra model was 83.6%. Further, a high value for the area under the receiver operating characteristic curve (0.958) was recorded. This study provides a basis for future studies on the investigation of myoglobin information associated with meat freshness.

Myoglobin and C-reactive protein are efficient and reliable early predictors of COVID-19 associated mortality.

Since the emergence of SARS-CoV-2, numerous studies have been attempting to determine biomarkers, which could rapidly and efficiently predict COVID-19 severity, however there is lack of consensus on a specific one. This retrospective cohort study is a comprehensive analysis of the initial symptoms, comorbidities and laboratory evaluation of patients, diagnosed with COVID-19 in Huoshenshan Hospital, Wuhan, from 4th February to 12th March, 2020. Based on the data collected from 63 severely ill patients from the onset of symptoms till the full recovery or demise, we found not only age (average 70) but also blood indicators as significant risk factors associated with multiple organ failure. The blood indices of all patients showed hepatic, renal, cardiac and hematopoietic dysfunction with imbalanced coagulatory biomarkers. We noticed that the levels of LDH (85%, P < .001), HBDH (76%, P < .001) and CRP (65%, P < .001) were significantly elevated in deceased patients, indicating hepatic impairment. Similarly, increased CK (15%, P = .002), Cre (37%, P = 0.102) and CysC (74%, P = 0.384) indicated renal damage. Cardiac injury was obvious from the significantly elevated level of Myoglobin (52%, P < .01), Troponin-I (65%, P = 0.273) and BNP (50%, P = .787). SARS-CoV-2 disturbs the hemolymphatic system as WBC# (73%, P = .002) and NEUT# (78%, P < .001) were significantly elevated in deceased patients. Likewise, the level of D-dimer (80%, P < .171), PT (87%, P = .031) and TT (57%, P = .053) was elevated, indicating coagulatory imbalances. We identified myoglobin and CRP as specific risk factors related to mortality and highly correlated to organ failure in COVID-19 disease.