Identification Of Endogenous Peptides Research Articles

Identification of endogenous peptides in maize

The extraction and identification of endogenous peptides from plants are relatively unexplored because of the degradation of peptides after extraction from plant tissues. In this study, we developed an optimized sample preparation protocol that combined constant water heating with plant protease inhibitors and TCA-acetone precipitation, resulting in the effective extraction of endogenous peptide from plants, such as maize, while diminishing unspecific protease activity. The results showed that a total of 2867 endogenous peptides were identified in three maize samples using this method, of which 2119 (73.9%) peptides were commonly identified in all three samples. The length and molecular weight of these peptides ranged from 8 to 25 amino acids and from 729.44 to 2980.62 Da, respectively, with 96.4% of the peptide scores greater than 40. These results indicated that our extraction method is highly reproducible, precise, and wide-ranging, providing optimized information for large-scale identification of plant endogenous peptides.

Identification of Endogenous Peptides in Nasal Swab Transport Media used in MALDI-TOF-MS Based COVID-19 Screening

Mass spectrometry (MS) based diagnostic detection of 2019 novel coronavirus infectious disease (COVID-19) has been postulated to be a useful alternative to classical PCR based diagnostics. These MS based approaches have the potential to be both rapid and sensitive and can be done on-site without requiring a dedicated laboratory or depending on constrained supply chains (i.e., reagents and consumables). Matrix-assisted laser desorption ionization (MALDI)–time-of-flight (TOF) MS has a long and established history of microorganism detection and systemic disease assessment. Previously, we have shown that automated machine learning (ML) enhanced MALDI-TOF-MS screening of nasal swabs can be both sensitive and specific for COVID-19 detection. The underlying molecules responsible for this detection are generally unknown nor are they required for this automated ML platform to detect COVID-19. However, the identification of these molecules is important for understanding both the mechanism of detection and potentially the biology of the underlying infection. Here, we used nanoscale liquid chromatography tandem MS to identify endogenous peptides found in nasal swab saline transport media to identify peptides in the same the mass over charge (m/z) values observed by the MALDI-TOF-MS method. With our peptidomics workflow, we demonstrate that we can identify endogenous peptides and endogenous protease cut sites. Further, we show that SARS-CoV-2 viral peptides were not readily detected and are highly unlikely to be responsible for the accuracy of MALDI based SARS-CoV-2 diagnostics. Further analysis with more samples will be needed to validate our findings, but the methodology proves to be promising.

An improved scoring method for the identification of endogenous peptides based on the Mascot MS/MS ion search.

To identify endogenous peptides using MS/MS analysis and searching against a polypeptide sequence database, a non-enzyme specific (NES) search considering all of the possible proteolytic cleavages is required. However, the use of a NES search generates more false positive hits than an enzyme specific search, and therefore shows lower identification performance. In this study, the use of the sub-ranked matches for improving the identification performance of the Mascot NES search was investigated and a new scoring method was developed that considered the contribution of all sub-ranked random match probabilities, named the contribution score (CS). The CS showed the highest identification sensitivity using the Mascot NES search with a full protein database when compared to the use of the Mascot first ranked score and the delta score (DS). The confident peptides identified by DS and CS were shown to be complementary. When applied to plant endogenous peptide identification, the identification numbers of tomato endogenous peptides using DS and CS were 176.3% and 184.2%, respectively, higher than the use of the first ranked score of Mascot. The combination of DS and CS identified 200.0% and 8.6% more tomato endogenous peptides compared to the use of Mascot and DS, respectively. This method by combining the CS and DS can significantly improve the identification performance of endogenous peptides without complex computational steps and is also able to improve the identification performance of the enzyme specific search. In addition to the application in the plant peptidomics analysis, this method may be applied to the improvement of peptidomics studies in different species. A web interface for calculating the DS and CS based on Mascot search results was developed herein.

Imaging and identification of endogenous peptides from rat pituitary embedded in egg yolk.

Mass spectrometry imaging (MSI) can provide accurate data containing the spatial distribution of endogenous peptides in tissue sections without previous treatment. One of the key issues in analyzing small samples is establishing a proper technique for mounting and manipulating collected tissue in order to avoid contamination of the sample with optimal cutting temperature (OCT) resin. We present a method for embedding rat pituitary tissue in a frozen egg yolk block, which enables its further imaging in experiments on a matrix-assisted laser desorption/ionization (MALDI) mass spectrometer with time-of-flight (TOF) analyzer. Embedding the sample in the egg yolk prevents contamination from the OCT resin, which decreases MALDI signal quality. In the present study we detected numerous m/z peaks related to endogenous peptides. We identified fifteen peptides and their post-translational modifications by tandem mass spectrometry (MS/MS) directly on tissue sections of the hypophysis posterior and intermediate lobes; among these peptides were vasopressin, oxytocin, copeptin, melanocyte-stimulating hormones and beta-endorphin. We also showed that egg yolk itself does not affect localization of peptides in the pituitary. Egg yolk embedding enables preparation of tissue sections from small tissue fragments to organs such as the pituitary gland, which is suitable for localization and identification of endogenous peptides by the MALDI-MSI and MALDI-MS/MS techniques.

Top‐down identification of endogenous peptides up to 9 kDa in cerebrospinal fluid and brain tissue by nanoelectrospray quadrupole time‐of‐flight tandem mass spectrometry

Recent work on protein and peptide biomarker patterns revealed the difficulties in identifying their molecular components, which is indispensable for validation of the biological context. Cerebrospinal fluid and brain tissue are used as sources to discover new biomarkers, e.g. for neurodegenerative diseases. Many of these biomarker candidates are peptides with a molecular mass of <10 kDa. Their identification is favourably achieved with a 'top-down' approach, because this requires less purification and an enzymatic cleavage will often not yield enough specific fragments for successful database searches. Here, we describe an approach using quadrupole time-of-flight mass spectrometry (TOFMS) as a highly efficient mass spectrometric purification and identification tool after off-line decomplexation of biological samples by liquid chromatography. After initial peptidomic screening with matrix-assisted laser desorption/ionization (MALDI) TOFMS, the elution behaviour in chromatography and the exact molecular mass were used to locate the same signals in nanoelectrospray measurements. Most of the peaks detected in MALDI-TOFMS could be retrieved in nanoelectrospray quadrupole TOFMS. Suitable collision energies for informative fragment spectra were investigated for different parent ions, charge states and molecular masses. After collision-induced dissociation, the resulting fragmentation data of multiply charged ions can become much more complicated than those derived from tryptic peptide digests. However, the mass accuracy and resolution of quadrupole TOF instruments results in high-quality data suitable for determining peptide sequences. The protein precursor, proteolytic processing and post-translational modifications were identified by automated database searches. This is demonstrated by the exemplary identifications of thymosin beta-4 (5.0 kDa) and NPY (4.3 kDa) from rat hypothalamic tissue and ubiquitin (8.6 kDa) from human cerebrospinal fluid. The high data quality should also allow for de novo identification. This methodology is generally applicable for peptides up to a molecular mass of about 10 kDa from body fluids, tissues or other biological sources.