Peptide Mass Fingerprinting Research Articles

Controlled Translocation of Proteins through a Biological Nanopore for Single-Protein Fingerprint Identification.

After the successful sequencing of nucleic acids, nanopore technology has now been applied to proteins. Recently, it has been demonstrated that an electro-osmotic flow can be used to induce the transport of unraveled polypeptides across nanopores. Polypeptide translocation, however, is too fast for accurate reading its amino acid compositions. Here, we show that the introduction of hydrophobic residues into the lumen of the nanopore reduces the protein translocation speed. Importantly, the introduction of a tyrosine at the entry of the nanopore and an isoleucine at the entry of the β-barrel of the nanopore reduced the speed of translocation to ∼10 amino acids/millisecond while keeping a relatively large ionic current, a crucial component for protein identification. These nanopores showed unique features within their current signatures, which may pave the way toward protein fingerprinting using nanopores.

Multi-proxy investigations of Bronze Age diet and environment in the hyper-arid eastern Tarim Basin (Lop Nur), northwest China

Humans have a long history of occupying ecologically marginal regions, and the ancient cultural mechanisms of adaptation to these challenging environments remain poorly understood. We examine archaeological deposits from a Bronze Age settlement in the hyper-arid eastern Tarim Basin (Lop Nur), identifying well-preserved plant remains and bone fragments that shed light on the palaeodiet and past environmental setting. We use the newly developed peptide mass fingerprinting method of ZooMS (zooarchaeology by mass spectrometry) and TimsTOF MS (trapped ion mobility time of flight mass spectrometry) to identify fragments of ancient fish bones as Cyprinidae and possible Salmonidae, indicating both their consumption and the former presence of intra-settlement fresh water-bodies. Remains of littoral and aquatic plants attest to a marshy palaeo-delta at the confluence of the Tarim and Kongque rivers, testifying to a former biodiverse oasis in the Lop Desert when humans occupied the site. We conclude that the use of both aquatic and desert peripheral resources, combined with the intentional management of local environments, were all essential prehistoric adaptations. In addition, we show that radiocarbon dates of aquatic remains have an offset spanning hundreds to thousands of years, due to a freshwater reservoir effect, requiring careful consideration of ages for archaeological bones in the broader region.

Identification, Purification, and Characterization of Kunitz-Type Protease Inhibitors from White-Fleshed Dragon Fruit (Selenicereus undatus) and Red-Fleshed Dragon Fruit (Selenicereus costaricensis) Seeds as Novel Food Allergens.

There have been clinical reports of allergies to dragon fruits. However, little is known about the allergens that trigger these reactions. This study aimed to investigate novel allergens in dragon fruits. The peptide mass fingerprints of two purified natural allergens were identified by LC-MS/MS with chymotrypsin proteolysis. The complete amino acid sequences of the allergens were deduced from cDNA amplicon sequences. The coding sequences of two allergens were inserted in the expression vector pColdI, and recombinant allergens were produced in Escherichia coli. Circular dichroism was performed to analyze the secondary structures of natural and recombinant allergens. Our results identified two novel allergens as Kunitz-type protease inhibitors. Recombinant allergens exhibited well-folded structures, predominantly composed of β-sheets, similar to their natural counterparts. IgE reactivity analysis with sera from ten patients primarily sensitized to dragon fruit indicated that Kunitz-type protease inhibitors are major allergens in dragon fruits. These results improve our understanding of allergen sources and provide important insights into the allergenicity of proteins from different species.

Identification and Activity Study of an Impurity Band Observed in the nrSDS-PAGE of Aflibercept.

Aflibercept is a biopharmaceutical targeting vascular endothelial growth factor (VEGF) that has shown promise in the treatment of neovascular age-related macular degeneration (nAMD) and diabetic macular edema (DME) in adults. Quality control studies of aflibercept employing non-reduced SDS-PAGE (nrSDS-PAGE) have shown that a significant variant band (IM1) is consistently present below the main band. Considering the quality control strategy of biopharmaceuticals, structural elucidation and functional studies are required. In this study, the variant bands in nrSDS-PAGE were collected through electroelution and identified by peptide mass fingerprinting based on liquid chromatography-tandem MS (LC-MS/MS). This variant was expressed using knob-into-hole (KIH) design transient transfection for the detection of ligand affinity, binding activity and biological activity. The variant band was formed by C-terminal truncation at position N99 of one chain in the aflibercept homodimer. Then, this variant was successfully expressed using KIH design transient transfection. The ligand affinity of the IM1 truncated variant was reduced by 18-fold, and neither binding activity nor biological activity were detected. The efficacy of aflibercept is influenced by the loss of biological activity of the variant. Therefore, this study supports the development of a quality control strategy for aflibercept.

Fixative or Finish? Identifying the Surface Coating on John Constable’s Drawings Using Peptide Mass Fingerprinting

ABSTRACT A survey of graphite drawings by the English painter John Constable revealed that in many cases the artist applied a coating overtop of the media. This thin, clear, glossy coating was characterized on over thirty drawings where it appeared to be the same material although employed in different ways. Several attempts were made to identify the material with minimal success until peptide mass fingerprinting (PMF) was used, and a minimally invasive sampling method involving vinyl eraser cubes proved to be a very easy and effective technique to collect sufficient sample material for the analysis. The results of testing samples from seven drawings conclusively identified the coating as isinglass, which Constable was using in other preparations of support materials for painting. As part of this work, putative markers for the identification of isinglass by PMF were developed.

Microfluidic Chip Coupled with MALDI-TOF MS for Multitarget Detection of Allergens in Crucian Carp (Carassius auratus).

The goal of the present study was to establish a rapid, simple method for simultaneous allergy testing of sera from multiple fish-allergic patients. Sera from fish-allergic patients were pooled and used for capturing allergens in fish muscle of crucian carp (Carassius auratus), which was studied as a fish model. Sarcoplasmic proteins of crucian carp (Carassius auratus) were extracted for the analysis of allergens. Anti-human IgE antibody-functionalized magnetic beads were utilized to collect IgE antibodies from human pooled sera. The isolation of allergenic proteins was immunomagnetically performed in microfluidic channels, and the elution of the captured allergenic proteins was done with 5% (v/v) acetic acid aqueous solution. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and peptide mass fingerprinting were used for the analysis of tryptic digests of eluted proteins. Ten potential allergenic proteins were identified from crucian carp (Carassius auratus). The present protocol provides a rapid, efficient, and simple method for simultaneous detection of multiple allergens, based on multitargeted antibodies from pooled sera of allergic patients. The constructed multiple antibody-modified MBs can be applied for the deallergenicity of food matrices. The efficiency of allergen detection can be greatly improved, with promising application in allergen discovery and filtration for other muscle-based foods.

Characteristics of Umami Taste of Soy Sauce Using Electronic Tongue, Amino Acid Analyzer, and MALDI-TOF MS.

The objective of this study was to investigate the umami characteristics of soy sauce using electronic tongue evaluation and amino acid composition and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. The soy sauce peptides were isolated from soy sauce using XAD-16 macroporous resin combined with ethanol solution. The results showed that the soy sauce peptide fraction eluted by 60% ethanol (SS-60%) exhibited a prominent umami taste, and the umami scores were highly positively correlated with the amino acid nitrogen contents of soy sauces. The umami scores of SS-60% were significantly positively correlated with the contents of free amino acids. Especially, Phe showed the highest positive correlation with the umami scores. In addition, five characteristic ion peaks with m/z at 499, 561, 643, 649, and 855 were identified in the peptide mass fingerprinting. Therefore, this study provides new insights into the umami characteristics for the taste evaluation and reality identification of soy sauce.

Impact of conjugation of whey protein concentrate with different carbohydrates: Monitoring structural and technofunctional variations

This study utilized whey protein concentrate (WPC) as the primary protein source, employing the Maillard reaction to link it with three distinct carbohydrates: lactose, maltodextrin, and gum Arabic. Mixtures were prepared with a 1:4 protein-to-carbohydrate ratio, and conjugation was performed using dry heating at a temperature of 60 °C, 50% relative humidity for 8 h processing time. Under the same conditions, native WPC was heated as a control group. The formation of conjugates was tracked by monitoring absorbance changes, browning index (BI), and reduction in free amino groups (FAG). All conjugates exhibited significant loss of FAG, correlating with an increase in BI. Shifts in protein fingerprint bands observed via Fourier Transform Infrared Spectroscopy suggested potential protein-carbohydrate interactions, while X-ray diffraction showed increased sample crystallinity post-conjugation. Solubility assessments across pH levels (3.0–8.0) indicated a 10–13% rise in solubility for conjugated samples compared to mixtures, broadening their potential applications. Moreover, the emulsion stability index demonstrated improved stability after conjugation. Overall, the Maillard reaction-mediated conjugation enhanced the solubility and stability of emulsions.

Amplifiable protein identification via residue-resolved barcoding and composition code counting.

Ultrasensitive protein identification is of paramount importance in basic research and clinical diagnostics but remains extremely challenging. A key bottleneck in preventing single-molecule protein sequencing is that, unlike the revolutionary nucleic acid sequencing methods that rely on the polymerase chain reaction (PCR) to amplify DNA and RNA molecules, protein molecules cannot be directly amplified. Decoding the proteins via amplification of certain fingerprints rather than the intact protein sequence thus represents an appealing alternative choice to address this formidable challenge. Herein, we report a proof-of-concept method that relies on residue-resolved DNA barcoding and composition code counting for amplifiable protein fingerprinting (AmproCode). In AmproCode, selective types of residues on peptides or proteins are chemically labeled with a DNA barcode, which can be amplified and quantified via quantitative PCR. The operation generates a relative ratio as the residue-resolved 'composition code' for each target protein that can be utilized as the fingerprint to determine its identity from the proteome database. We developed a database searching algorithm and applied it to assess the coverage of the whole proteome and secretome via computational simulations, proving the theoretical feasibility of AmproCode. We then designed the residue-specific DNA barcoding and amplification workflow, and identified different synthetic model peptides found in the secretome at as low as the fmol/L level for demonstration. These results build the foundation for an unprecedented amplifiable protein fingerprinting method. We believe that, in the future, AmproCode could ultimately realize single-molecule amplifiable identification of trace complex samples without further purification, and it may open a new avenue in the development of next-generation protein sequencing techniques.

Plasma-based proteomic profiling identifies the distinct regulation of proteins in hyperplasia and endometrial cancer

BackgroundAmong gynaecological malignancies, endometrial cancer (EC) is the most prevalent type of uterine cancer affecting women. This study explored the proteomic profiles of plasma samples obtained from EC patients, those with hyperplasia (Hy), and a control group (CO). A combination of techniques, such as 2D-DIGE, mass spectrometry, and bioinformatics, including pathway analysis, was used to identify proteins with modified expression levels, biomarkers and their associated metabolic pathways in these groups.MethodsThirty-four patients, categorized into three groups—10 with EC, 12 with Hy, and 12 CO—between the ages of 46 and 75 years old were included in the study. Untargeted proteomic analysis was carried out using two-dimensional difference in gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).ResultsIn all three groups, 114 proteins that were significantly (p ≤ 0.05 and fold change ≥ 1.5) altered were successfully identified using peptide mass fingerprints (PMFs). Compared with those in the control group (CO), the EC samples had 85 differentially expressed proteins (39 upregulated and 46 downregulated), and in the Hy group, 81 proteins were dysregulated (40 upregulated and 41 downregulated) compared to those in the CO group, while 33 proteins exhibited differential regulation (12 upregulated and 21 downregulated) in the EC plasma samples compared to those in the Hy group. Vitamin D binding protein and complement C3 distinguished Hy and EC from CO with the greatest changes in expression. Among the differentially expressed proteins identified, enzymes with catalytic activity represented the largest group (42.9%). In terms of biological processes, most of the proteins were involved in cellular processes (28.8%), followed by metabolic processes (16.7%). STRING analysis for protein interactions revealed that the significantly differentially abundant proteins in the three groups are involved in three main biological processes: signalling of complement and coagulation cascades, regulation of insulin-like growth factor (IGF) transport and uptake by insulin-like growth factor binding proteins (IGFBPs), and plasma lipoprotein assembly, remodelling, and clearance.ConclusionThe identified plasma protein markers have the potential to serve as biomarkers for differentiating between EC and Hy, as well as for early diagnosis and monitoring of cancer progression.

Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry Combined with Chemometrics for Protein Profiling and Classification of Boiled and Extruded Quinoa from Conventional and Organic Crops

Quinoa is an Andean crop that stands out as a high-quality protein-rich and gluten-free food. However, its increasing popularity exposes quinoa products to the potential risk of adulteration with cheaper cereals. Consequently, there is a need for novel methodologies to accurately characterize the composition of quinoa, which is influenced not only by the variety type but also by the farming and processing conditions. In this study, we present a rapid and straightforward method based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to generate global fingerprints of quinoa proteins from white quinoa varieties, which were cultivated under conventional and organic farming and processed through boiling and extrusion. The mass spectra of the different protein extracts were processed using the MALDIquant software (version 1.19.3), detecting 49 proteins (with 31 tentatively identified). Intensity values from these proteins were then considered protein fingerprints for multivariate data analysis. Our results revealed reliable partial least squares-discriminant analysis (PLS-DA) classification models for distinguishing between farming and processing conditions, and the detected proteins that were critical for differentiation. They confirm the effectiveness of tracing the agricultural origins and technological treatments of quinoa grains through protein fingerprinting by MALDI-TOF-MS and chemometrics. This untargeted approach offers promising applications in food control and the food-processing industry.

Tandam mass spectrometry instrumentation and application in pharmaceutical analysis

Proteins and peptides can be analyzed using mass spectrometry (MS) using a range of techniques, including matrix-aided laser desorption ionization-mass spectrometry (MALDI-MS) and electrospray ionization-mass spectrometry (ESI-MS). These techniques make it possible to determine a protein's mass as an intact molecule or to identify a protein using peptide-mass fingerprinting that is produced during enzymatic digestion. The amino acid sequence of proteins (top-down and middle-down proteomics) and peptides (bottom-up proteomics) can be ascertained by fragmenting the proteins and peptides using tandem mass spectrometry (MS/MS). Furthermore, post-translational modifications (PTMs) of proteins and peptides can be identified using tandem mass spectrometry. In this article, we go over the use of MS/MS in biomedical research and provide concrete examples of how to identify proteins, peptides, and their PTMs as useful biomarkers for diagnosis and treatment. In numerous applications, tandem mass spectrometry (MS/MS) has shown to be a practical and efficient analytical method for the direct detection of target compounds in food samples. It combines the power of MS/MS as an identification and confirmation approach with the separation capabilities of chromatography when used with chromatographic techniques.

Finding the right plaice at the right time: Multi‐molecular analysis of flatfish reveals historical catch habitats

AbstractFlatfish are ecologically diverse species that commonly occur in marine environments, but also in estuarine and riverine habitats. This complicates the examination of the potential role of flatfish in the ‘marine fish event horizon’, an economic shift in human exploitation from freshwater to marine fish species during the 10–11th centuries CE around the southern North Sea. This study represents the first multi‐disciplinary investigation of flatfish remains to make species‐specific interpretations of flatfish exploitation. Peptide mass fingerprinting and multi‐isotope analysis of carbon (δ13C), nitrogen (δ15N) and sulphur (δ34S) was performed on collagen from 356 archaeological flatfish and 120 comparative archaeological marine or freshwater species to explore the catch habitat of individual flatfish species between 600 and 1600 CE from the North Sea area. European flounder show signals reflecting both freshwater and marine environments, while other flatfish show only those of marine habitats. A subtle shift towards more marine exploitation towards the end of the period is identified, corresponding to the observed transition in targeted species from flounder to plaice throughout the medieval period. Sites show slight differences in δ13C and δ34S within the same species, related to the local environments. Remarkable is the high abundance of marine plaice and flounder during the early medieval period, which shows clear marine or coastal exploitation of flatfish early on, well before the previously accepted onset of the marine fish event horizon. This indicates a gradual shift from coastal to open marine fish exploitation over the medieval period.

Proteome analysis, genetic characterization, and antibiotic resistance patterns of Klebsiella pneumoniae clinical isolates

Klebsiella pneumoniae (K. pneumoniae) is a member of the ESKAPE group and is responsible for severe community and healthcare-associated infections. Certain Klebsiella species have very similar phenotypes, which presents a challenge in identifying K. pneumoniae. Multidrug-resistant K. pneumoniae is also a serious global problem that needs to be addressed. A total of 190 isolates were isolated from urine (n = 69), respiratory (n = 52), wound (n = 48) and blood (n = 21) samples collected from various hospitals in the Al-Qassim, Saudi Arabia, between March 2021 and October 2022. Our study aimed to rapidly and accurately detect K. pneumoniae using the Peptide Mass Fingerprinting (PMF) technique, confirmed by real-time PCR. Additionally, screening for antibiotic susceptibility and resistance was conducted. The primary methods for identifying K. pneumoniae isolates were culture, Gram staining, and the Vitek® 2 ID Compact system. An automated MALDI Biotyper (MBT) instrument was used for proteome identification, which was subsequently confirmed using SYBR green real-time polymerase chain reaction (real-time PCR) and microfluidic electrophoresis assays. Vitek® 2 AST-GN66 cards were utilized to evaluate the antimicrobial sensitivity of K. pneumoniae isolates. According to our results, Vitek® 2 Compact accurately identified 178 out of 190 (93.68%) K. pneumoniae isolates, while the PMF technique correctly detected 188 out of 190 (98.95%) isolates with a score value of 2.00 or higher. Principal component analysis was conducted using MBT Compass software to classify K. pneumoniae isolates based on their structure. Based on the analysis of the single peak intensities generated by MBT, the highest peak values were found at 3444, 5022, 5525, 6847, and 7537 m/z. K. pneumoniae gene testing confirmed the PMF results, with 90.53% detecting entrobactin, 70% detecting 16 S rRNA, and 32.63% detecting ferric iron uptake. The resistance of the K. pneumoniae isolates to antibiotics was as follows: 64.75% for cefazolin, 62.63% for trimethoprim/sulfamethoxazole, 59.45% for ampicillin, 58.42% for cefoxitin, 57.37% for ceftriaxone, 53.68% for cefepime, 52.11% for ampicillin-sulbactam, 50.53% for ceftazidime, 52.11% for ertapenem, and 49.47% for imipenem. Based on the results of the double-disk synergy test, 93 out of 190 (48.95%) K. pneumoniae isolates were extended-spectrum beta-lactamase. In conclusion, PMF is a powerful analytical technique used to identify K. pneumoniae isolates from clinical samples based on their proteomic characteristics. K. pneumoniae isolates have shown increasing resistance to antibiotics from different classes, including carbapenem, which poses a significant threat to human health as these infections may become difficult to treat.

Inferring molecular inhibition potency with AlphaFold predicted structures

Even though in silico drug ligand-based methods have been successful in predicting interactions with known target proteins, they struggle with new, unassessed targets. To address this challenge, we propose an approach that integrates structural data from AlphaFold 2 predicted protein structures into machine learning models. Our method extracts 3D structural protein fingerprints and combines them with ligand structural data to train a single machine learning model. This model captures the relationship between ligand properties and the unique structural features of various target proteins, enabling predictions for never before tested molecules and protein targets. To assess our model, we used a dataset of 144 Human G-protein Coupled Receptors (GPCRs) with over 140,000 measured inhibition constants (Ki) values. Results strongly suggest that our approach performs as well as state-of-the-art ligand-based methods. In a second modeling approach that used 129 targets for training and a separate test set of 15 different protein targets, our model correctly predicted interactions for 73% of targets, with explained variances exceeding 0.50 in 22% of cases. Our findings further verified that the usage of experimentally determined protein structures produced models that were statistically indistinct from the Alphafold synthetic structures. This study presents a proteo-chemometric drug screening approach that uses a simple and scalable method for extracting protein structural information for usage in machine learning models capable of predicting protein-molecule interactions even for orphan targets.

Minimally Invasive Approaches to Diagnose and Monitor Eosinophilic GI Diseases.

This review seeks to understand novel avenues for eosinophilic GI disease management. Biomarkers offer a unique and non-invasive approach to tracking EoE disease progression. While no biomarkers have definitively met the diagnostic criteria for eosinophilic GI diseases, some biomarkers have been shown to be associated with disease activity. Here, we examine the potential of recently studied biomarkers. Current research shows advancements in blood, luminal fluid, and breath testing. Particular areas of interest include mRNA analyses, protein fingerprinting, amplicon sequence variants (ASVs), T cells and IgE receptors, eosinophilic cationic proteins, cytokines, and nitric oxide exhalation. Preliminary results showed that mucosal biomarkers, directly captured from the esophagus, may reflect the best representation of biopsy-based results, in contrast to biomarkers obtained from indirect or peripheral (blood, breath) methods. However, this is based on limited clinical studies without sufficient numbers to evaluate true diagnostic accuracy. Large-scale randomized trials are needed to fully ascertain both the optimal sampling technique and the specific biomarkers that reflect diagnostic status of the disease.

Topographic fingerprinting of single proteins and proteoforms.

Topographic fingerprinting of single proteins and proteoforms.

A promoted MALDI-TOF-MS platform towards rapid and accurate identifications of bacteria

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is emerging as the most powerful tool for the identification of bacteria. Herein, we demonstrated that the capability of MALDI-TOF-MS towards the discrimination of clinical bacteria can be further dramatically improved. A cooperative matrix consisting of (E)-propyl α-cyano-4-hydroxylcinnamylate (CHCA-C3) and α-cyano-4-hydroxycinnamic acid (CHCA) was used to replace the commonly used matrix CHCA. As a result, 1.4–3.8 times more fingerprint peaks have been identified by the cooperative matrix in comparison with CHCA when six different bacteria were studied. The peaks distribute in the wider m/z range, and the spectral quality has also been significantly improved. As an example of practical applications, the direct and more reliable discrimination between Escherichia coli (E. coli) and Shigella sonnei (S. sonnei) was achieved based on their protein fingerprints, which was previously claimed to be impossible by CHCA. Thus, a promoted MALDI-TOF-MS platform towards bacterial identification has been established by utilizing the new cooperative matrix, which will exhibit great potential for many applications.

Rapid detection and quantification of milk adulteration using MALDI-MS protein profiling and multivariate calibration

The adulteration of high-value milks with low-value milks is a common fraudulent practice in the dairy industry. This not only results in economic or quality prejudice, but also poses a potential threat to individuals with sensitivities or allergies. Profiling the major whey and casein milk proteins can be a crucial tool in combating this malpractice, given that each mammal species exhibits a unique protein fingerprint. In this study, we employed matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-MS) as a rapid and straightforward method for profiling proteins from bovine, caprine, and ovine milks. Subsequently, we analyzed binary mixtures of goat and sheep milks with bovine milk to establish multivariate calibration models using partial least-squares (PLS) to quantify the adulteration of goat and sheep milks. Proteins were identified according to their molecular mass (Mr) and their intensities were considered as multivariate data. To achieve satisfactory calibration and validation results, normalization through variable augmentation was necessary, enabling the quantification of bovine milk across the entire adulteration range. This uncomplicated approach based on MALDI-MS protein profiling and PLS shows significant potential for qualitative and quantitative assessments of milk adulteration.

Full-length single-molecule protein fingerprinting.

Proteins are the primary functional actors of the cell. While proteoform diversity is known to be highly biologically relevant, current protein analysis methods are of limited use for distinguishing proteoforms. Mass spectrometric methods, in particular, often provide only ambiguous information on post-translational modification sites, and sequences of co-existing modifications may not be resolved. Here we demonstrate fluorescence resonance energy transfer (FRET)-based single-molecule protein fingerprinting to map the location of individual amino acids and post-translational modifications within single full-length protein molecules. Our data show that both intrinsically disordered proteins and folded globular proteins can be fingerprinted with a subnanometer resolution, achieved by probing the amino acids one by one using single-molecule FRET via DNA exchange. This capability was demonstrated through the analysis of alpha-synuclein, an intrinsically disordered protein, by accurately quantifying isoforms in mixtures using a machine learning classifier, and by determining the locations of two O-GlcNAc moieties. Furthermore, we demonstrate fingerprinting of the globular proteins Bcl-2-like protein 1, procalcitonin and S100A9. We anticipate that our ability to perform proteoform identification with the ultimate sensitivity may unlock exciting new venues in proteomics research and biomarker-based diagnosis.