ABSTRACT
In this work, we have extended our previously proposed approach for determining protein concentrations in human serum (using MALDI-TOF mass spectrometry) to include simultaneous analysis of several proteins associated with acute inflammation (alpha-2-macroglobulin, fetuin-A, serum amyloid A1). This technique can be used to diagnose systemic inflammation and provides results in 4-5 h. The developed approach was verified using standard immunological methods (ELISA). Samples from 87 individuals, in specific groups, were used for testing and validation: control; inflammatory soft tissue disease accompanied by sepsis; influenza A infection; or COVID-19. The feasibility of differentiating patient groups with the aforementioned conditions was analyzed using a combination of the inflammatory markers described. For fetuin-A and serum amyloid A1, diagnostically significant concentration ranges were established.
Subject(s)
COVID-19 , Biomarkers , Humans , SARS-CoV-2 , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
One of the main functions of alpha-2-macroglobulin (A2M) in human blood serum is the binding of all classes of protease. It is known that trypsin, after such interaction, possesses modified proteolytic activity. Trypsin first hydrolyzes two bonds in A2M's 'bait region', and the peptide 705VGFYESDVMGR715 is released from A2M. In this work, specifics of the A2M-trypsin interaction were used to determine A2M concentration directly in human blood serum using MALDI mass-spectrometry. Following exogenous addition of trypsin to human blood serum in vitro, the concentration of the VGFYESDVMGR peptide was measured, using its isotopically-labeled analogue (18O), and A2M concentration was calculated. The optimized mass spectrometric approach was verified using a standard method for A2M concentration determination (ELISA) and the relevant statistical analysis methods. It was also shown that trypsin's modified proteolytic activity in the presence of serum A2M can be used to analyze other serum proteins, including potential biomarkers of pathological processes. Thus, this work describes a promising approach to serum biomarker analysis that can be technically extended in several useful directions.
Subject(s)
Peptides/blood , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Trypsin/blood , Biomarkers/blood , Humans , alpha-MacroglobulinsABSTRACT
Amino acid analysis is among the most accurate methods for absolute quantification of proteins and peptides. Here we combine acid hydrolysis with the addition of isotopically labeled standard amino acids and analysis by mass spectrometry for accurate and sensitive protein quantitation. Quantitation of less than 10 fmol of protein standards with errors below 10% has been demonstrated using this method.
Subject(s)
Amino Acids/analysis , Proteins/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Amino Acids/chemistry , Carbon Isotopes/chemistry , Chromatography, High Pressure Liquid/methods , Hydrolysis , Nitrogen Isotopes/chemistry , Proteins/chemistryABSTRACT
The fibrillogenesis of a peptide corresponding to residues 35-51 of human α-lactalbumin (¹GYDTQAIVENNESTEYG¹7) can be dramatically enhanced by the addition of a tetrapeptide TDYG homologous to its C-terminus (TEYG). Generation of spontaneous hydrolytic products similar to this peptide was demonstrated by mass-spectrometry analysis of GYDTQAIVENNESTEYG peptide solution components during fibrillogenesis. Possible mechanisms and roles of short peptides in protein metabolism are discussed.
Subject(s)
Amyloid/genetics , Mutation , Amino Acid Sequence , Amyloid/chemistry , Amyloid/ultrastructure , Humans , Microscopy, Electron, Transmission , Molecular Sequence Data , ProteolysisABSTRACT
Amino acid analysis is among the most accurate methods for absolute quantification of proteins and peptides. Here, we combine acid hydrolysis with the addition of isotopically labeled standard amino acids and analysis by mass spectrometry for accurate and sensitive protein quantitation. Quantitation of less than 10 fmol of protein standards with errors below 10% has been demonstrated using this method.
Subject(s)
Acids/metabolism , Amino Acids/analysis , Mass Spectrometry/methods , Proteins/analysis , Amino Acids/chemistry , Hydrolysis , Proteins/chemistryABSTRACT
Chronic imbalance between production and degradation of the human amyloid-beta peptide (Abeta) is assumed to play an important role in pathogenesis of Alzheimer's disease (AD). Post-translational modifications of Abeta could influence its interactions with specifically cleaving proteases and, therefore, perturb the Abeta homeostasis. The angiotensin-converting enzyme (ACE) was previously shown to degrade non-modified Abeta in vitro and in cells. In the presented work, we investigated the effect of isomerization of Asp-7, a common non-enzymatic age-related modification found in AD-associated Abeta species, on hydrolysis of Abeta by ACE. Two synthetic peptides corresponding to the Abeta region 1-16 with either Asp or isoAsp residues in position 7 were examined as monomeric soluble substrates for the N- as well as for the C-domain of ACE. The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) coupled with the (18)O-labeled internal standard approach has allowed us to show that (i) the N-domain of ACE (N-ACE), but not the C-domain, selectively cleaves the Arg-5-His-6 bond in both peptides, and that (ii) N-ACE hydrolyzes the isoAsp-7 analogue more efficiently than the non-modified one. Our results demonstrate a new endopeptidase activity of N-ACE as well as high preference of the domain to recognize and hydrolyze the isomerized Abeta species that were earlier suggested to promote AD pathogenesis. The results suggest the need for further analysis of biological effects of isomerized Abeta and its interaction with ACE in AD pathogenesis.
Subject(s)
Amyloid beta-Peptides/chemistry , Arginine/chemistry , Histidine/chemistry , Peptide Fragments/chemistry , Peptidyl-Dipeptidase A/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Alzheimer Disease/enzymology , Alzheimer Disease/etiology , Humans , Hydrolysis , IsomerismABSTRACT
A series of relatively short (GCC)(n) triplet repeats (n = 3-30) located within regulatory regions of many mammalian genes may be considered as putative cis-acting transcriptional elements (GCC-elements). Fragile X-mental retardation syndrome is caused by an expansion of (GCC)(n) triplet repeats within the 5'-untranslated region of the human fragile X-mental retardation 1 (FMR1) gene. The present study aimed to characterize a novel human (GCC)(n)-binding protein and investigate its possible role in the regulation of the FMR1 gene. A novel human (GCC)(n)-binding protein, p56, was isolated and identified as a Krüppel-like transcription factor, ZF5, by MALDI-TOF analysis. The capacity of ZF5 to specifically interact with (GCC)(n) triplet repeats was confirmed by the electrophoretic mobility shift assay with purified recombinant ZF5 protein. In cotransfection experiments, ZF5 overexpression repressed activity of the GCC-element containing mouse ribosomal protein L32 gene promoter. Moreover, RNA interference assay results showed that endogenous ZF5 acts as a repressor of the human FMR1 gene. Thus, these data identify a new class of ZF5 targets, a subset of genes containing GCC-elements in their regulatory regions, and raise the question of whether transcription factor ZF5 is implicated in the pathogenesis of fragile X syndrome.
Subject(s)
Down-Regulation , Fragile X Mental Retardation Protein/genetics , Kruppel-Like Transcription Factors/metabolism , Repressor Proteins/metabolism , Amino Acid Sequence , Base Sequence , Cell Line, Tumor , Humans , Kruppel-Like Transcription Factors/chemistry , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/isolation & purification , Molecular Weight , Nuclear Proteins/isolation & purification , Nuclear Proteins/metabolism , Promoter Regions, Genetic/genetics , Protein Binding , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Repetitive Sequences, Nucleic Acid/genetics , Repressor Proteins/chemistry , Repressor Proteins/genetics , Repressor Proteins/isolation & purification , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Quantitation by mass spectrometry is increasingly used to monitor protein levels in biological samples. Most of the current methods are based on the relative comparison of protein quantities but are not suited for the determination of the absolute amount of a given protein. Here we describe a method for the absolute quantitation of proteins that is based on amino acid analysis by matrix-assisted laser desorption/ionization mass spectrometry. Proteins are completely hydrolyzed by acid hydrolysis and then mixed with standards of isotopically labeled amino acids. For the presented study, lysine, leucine, and two different types of labeled arginine were examined as standards. Quantitation of proteins is then achieved by measuring the ratios of labeled and unlabeled amino acids. The method has a sensitivity down to the low-femtomole range and can be applied to quantitate proteins separated by gel electrophoresis. Furthermore, we demonstrate that a mixture of two proteins can be quantitated using two labeled amino acids simultaneously.