The peptide-to-protein counts of serum samples were previously statistically analyzed using the generic statistical system S [ 8 ] or SAS [ 28 , 30 , 31 , 33 , 34 ] but in this study the data was analyzed using the generic open-source R statistical system [ 37 ]. The distributions and quantile plots of the peptide-to-protein counts, log10 intensity, expectation values E , peptide mass and delta mass values calculated by X!
The peptide p-values from X! The probability distribution of proteins over Gene Ontology GO terms, as well as their protein interactions from the Kyoto Encyclopedia of Genes and Genomes KEGG protein interactions were calculated with respect to random expectation using the corrections provided by Bonferroni, or Benjamini and Hochberg [ 42 ] for the number of hypotheses tested.
The best fit peptides from X! The X! Furthermore, X! TANDEM identified many cellular proteins from endogenous peptides in EDTA plasma from the ion trap that showed good agreement with the proteins from exogenous tryptic digestion of blood fluids and in agreement with previous results of endogenous peptides from the Paul 3D ion trap [ 3 ].
After discounting apparently spurious proteins from the negative controls of random, noise solvents alone or typically digested dust controls, the many cellular proteins identified by X! TANDEM algorithm was selected to examine the proteins in plasma on ice or at room temperature since it directly computes a p-value that the protein detected was merely random chance and has been compared to that of random spectra or noise for fully tryptic peptides from exogenous digestion [ 28 , 29 , 30 , 31 ].
One method to remove these redundant correlations to more than one protein accession number is to select the accession number with the highest number of correlations per gene symbol and then analyze the data at the peptide and protein level to yield a list of distinct RANK1 peptides. There were many more high-intensity peptides observed in the room temperature versus the preserved EDTA plasma Fig.
The list of Gene Symbols identified with the cumulative p-value and the FDR corrected q-value are provided in the Additional file 1 , Additional file 2 , Additional file 3 , Additional file 4. The redundant peptide correlations from X! The Filtered Filter 2 data can be found in Additional file 1 , Additional file 2 , Additional file 3 , Additional file 4.
TANDEM provided robust statistical reliability in agreement with previous results [ 28 , 29 , 30 , 31 , 32 ]. Accepting only protein accessions with 5 or more independent peptides resulted in 28, correlations. Proteins with at least 5 peptides from X! Plasma has been shown to express a net weak tryptic activity towards exogenous protein substrates [ 45 ].
New peptides, and larger numbers of peptides, were observed from plasma proteins incubated at room temperature including many cellular proteins such as G-proteins, dyneins, myosin and actin associated filament proteins, kininogen, signalling enzymes such as small G proteins and other factors, such as nucleic acid binding proteins among many others Fig. The filtered proteins of human plasma frozen or maintained on ice from at least 5 fully tryptic endogenously cleaved peptides as correlated by the X!
The set of proteins sequences with 5 independent peptides was collapsed by STRING to a set of Gene Symbols that after manual annotation were examined in detail using the R generic statistical analysis system. The STRING algorithm looks for previously established relations between the set of identified proteins [ 35 , 36 , 43 ]. The STRING algorithm tests to determine if the data set is merely a random assemblage of proteins or a network of proteins that show structural and functional relationships and so are known to exist and function together.
The complete network derived from at least three peptides from X! Proteins associated with membrane bound vesicles, secretion, exocytosis and exosomes were also observed. Proteins observed in cells as well as from specific tissues including the ovary and internal female genital organs, the colorectum and large intestine, adrenal gland, spinal cord, gall bladder, heart, urinary bladder, and blood plasma among others were enriched not shown.
Blood plasma was found to be enriched in peptides from proteins previously associated with disease, including monogenic, anatomical, autosomal, nervous system, musculoskeletal, recessive and sensory system disease not shown. Random mis-correlations from SEQUEST to giant proteins, noise and dust contamination are likely shared by all treatments to a roughly equal amount.
Since false positive identifications should tend to be roughly equal in the room temperature versus ice cold samples, imposing a fivefold increase with temperature should tend to remove false positive identifications that should be common to both conditions. The protein interaction network of proteins from EDTA plasma that was incubated at room temperature detected from at least 5 fully tryptic endogenous peptides by the X!
High resolution and low resolution mass spectrometers have been compared for the analysis of peptides from human blood fluids that showed good agreement but the ion trap is more sensitive and more robust [ 3 , 4 , 5 , 7 , 8 ]. High resolution mass spectrometers are required for the use of accurate mass strategy [ 46 ], for the use of isotopic [ 47 , 48 ] or isobaric strategies [ 49 ] or carbohydrate analysis [ 50 ].
Simple ion traps are sufficient for the identification of tryptic peptides from Eukaryotes and so are appropriate for the sampling of peptides from human samples that have been fractioned by chromatography [ 51 , 52 , 53 ]. The low error rate of the sensitive ion trap for proteins with multiple peptides is well established and shows that confidence builds with the number of peptides identified such that proteins with three or more independent peptides show a low error rate [ 28 , 29 , 30 , 31 , 54 , 55 ].
Moreover, proteins identified by low resolution mass spectrometers have been confirmed by Western blots, siRNA, drugs, immune cyto staining or GFP fusions [ 3 , 37 , 38 ]. However, given the low p-values presented here, the corrected q-values from the FDR calculated by the method of Benjamini and Hochberg [ 42 ] were highly significant. The aim of this study was to determine the nature of the proteins and protein complexes that are susceptible to degradation by comparing endogenous tryptic peptides from normal human EDTA plasma on ice versus plasma incubated at room temperature.
Plasma has a net mild tryptic activity [ 45 ] but also shows both C and N terminal exopeptidase activity [ 8 , 58 ] that might confound the results of tryptic correlations. It is well established that C and N terminal exopeptidases are active in blood plasma and these enzymes cleave amino acids from both ends of tryptic peptides [ 8 ].
In agreement with previous statistical analysis, the results of X! Thus, to provide a more complete list of plasma peptides for the Additional file 1 , Additional file 2 , Additional file 3 , Additional file 4 that also limits error, we have selected the proteins identified by SEQUEST with at least 5 peptides where the proteins were also detected by X!
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Collapsing the identical peptides observed in homologous proteins to one representative gene symbol is a convenient way to represent proteomic data that reduces the redundancy from proteins that share overlapping amino acid sequences. The analysis of the results stored in SQL Server can be computed on a per Gene Symbol basis using R to eliminate redundancy from similar protein sequences. Previously we collapsed these multiple proteins into protein types by manual annotation [ 7 ], or mapping short sequences into longer representative sequences using SQL or BLAST [ 7 , 44 ]. We conclude that caution must be exercised when analyzing proteins on these known contaminant lists using SEQUEST to ensure the frequency of detection is much higher than background.
In agreement with previous results, the measured type I error rate of fully tryptic peptides from X! TANDEM was low and so is one means to assign identity to low abundance endogenous tryptic peptides from plasma with confidence [ 28 , 29 ].
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Here, the use of a physical noise filter of E3 counts, together with the significant fit of at least three peptides [ 55 ] by X! TANDEM led to the apparent identification of many cellular proteins in plasma that are provided in the Additional file 1 , Additional file 2 , Additional file 3 , Additional file 4. TANDEM algorithm indicates that a pool of cellular proteins, with some major plasma proteins, are degraded in plasma with incubation at room temperature.
The results indicate that the cellular proteins released into plasma are not as stable as the major plasma proteins. The cumulative p-values and FDR corrected q-values from X! TANDEM indicate that peptides from types of functionally or structurally related proteins [ 7 , 44 ] can be accurately identified by a one-step C18 chromatography preparation.
The STRING algorithm revealed a complex set of relationships at the level of homology, transcriptional regulation, protein—protein interactions, protein families and signal pathways. In agreement with the significant p-values assigned by the X!
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Structural, functional and protein-interactions were revealed in the relationships between many of the degraded proteins of human plasma and a variety of cellular factors were observed that may have great biological significance. Cytoskeleton associated Ankyrin itself from red blood cells has been previously shown to encode an activity that results in the cleavage of complement [ 60 ]. A significantly large number of reciprocal protein—protein interactions were observed between the set of proteins identified that essentially precludes high rates of false positive identification, and clearly indicates that some circulating protein complexes are susceptible to degradation by endogenous tryptic peptide activities soon after incubation at room temperature.
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One simply strategy to avoid potential false positive correlations is to take the ratio of the room temperature where tryptic proteases may act versus ice cold samples where tryptic protease activity is significantly reduced thus largely removing the results of random mis-correlations and noise shared by both. TANDEM algorithms results in a list of cellular proteins that are cleaved in plasma at room temperature. Since the apparently spurious mis-correlations to titin TTN or other giant proteins from SEQUEST are shared by all treatments, one strategy to avoid noise and mis-correlation from the sensitive SEQUEST algorithm is to select tryptic peptides that are specific to room temperature incubation thereby largely eliminating the bias towards erroneous identifications to giant proteins.
Quantifying the endogenous peptides may represent a direct approach to monitor levels of cellular protein in the plasma between experimental treatments or physiological states while avoiding the interfering peptides from albumin, apolipoproteins and other common plasma molecules [ 7 ]. Peptides from KNG1 were observed at room temperature and it is known to play a role in the regulation of the complement cascade [ 61 ].
Alternative splicing of the KNG1 transcript generates the cysteine protease inhibitor, high-molecular-weight kininogen HMWK , that functions in the regulation of plasminogen that activate s the complement system and blood coagulation, and in turn is cleaved by the enzyme kallikrein to produce bradykinin that regulates inflammatory responses [ 62 ].
Kininogen and Ankyrin have been previously observed from tryptic peptides from serum [ 7 ] and in degraded plasma samples [ 27 ]. TANDEM or by taking the ratio of room temperature versus preserved samples that was supported by the comparison to the null model of random peptides and the non-random distribution across GO terms. The agreement between independent algorithms, the p-value of the peptides generated in X! The simplest model that explains all of the data collected here and elsewhere is that the major proteins of plasma such as albumin and apolipoproteins are relatively stable in plasma but that complexes of cellular proteins in circulation are more susceptible to degradation.
The ex vivo artefacts of tryptic peptides released by endogenous endopeptidases may be used to identify and quantify plasma proteins. Understanding and documenting the set of peptides cleaved from proteins that appear over the baseline immediately after sample collection is a pre-condition for the rational exploration of blood peptides across disease or other physiological states.
Collecting EDTA plasma directly on ice essentially prevents the degradation of blood proteins. Putnam F. The plasma proteins: structure function, and genetic control. New York: Academic Press; Tietz fundamentals of clinical chemistry. Philadelphia: Saunders; Tucholska M, et al.
tax-marusa.com/order/map21.php J Proteomics. Anal Biochem. Williams D, et al. Precipitation and selective extraction of human serum endogenous peptides with analysis by quadrupole time-of-flight mass spectrometry reveals posttranslational modifications and low-abundance peptides. Tools Request permission Export citation Add to favorites Track citation.
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