Extraction of naturally occurring peptides versus the tryptic digestion of proteins from fetal versus adult bovine serum for LC-ESI-MS/MS.

The naturally occurring peptides and digested proteins of fetal versus adult bovine serum were compared by LC-ESI-MS/MS after correction against noise from blank injections and random MS/MS spectra as statistical controls. Serum peptides were extracted by differential precipitation with mixtures of acetonitrile and water. Serum proteins were separated by partition chromatography over quaternary amine resin followed by tryptic digestion. The rigorous X!TANDEM goodness of fit algorithm that has a low error rate as demonstrated by low FDR q-values (q ≤ 0.01) showed qualitative and quantitative agreement with the SEQUEST cross correlation algorithm on 12,052 protein gene symbols. Tryptic digestion provided a quantitative identification of the serum proteins where observation frequency reflected known high abundance. In contrast, the naturally occurring peptides reflected the cleavage of common serum proteins such as C4A, C3, FGB, HPX, A2M but also proteins in lower concentration such as F13A1, IK, collagens and protocadherins. Proteins associated with cellular growth and development such as actins (ACT), ribosomal proteins like Ribosomal protein S6 (RPS6), synthetic enzymes and extracellular matrix factors were enriched in fetal calf serum. In contrast to the large literature from cord blood, IgG light chains were absent from fetal serum as observed by LC-ESI-MS/MS and confirmed by ELISA.

LEDGF is a new growth factor in fetal serum.

Fetal serum supports the immortal growth of mammalian cell lines in culture while adult serum leads to the terminal differentiation and death of cells in culture. Many of the proteins in fetal serum that support the indefinite division and growth of cancerous cell lines remain obscure. The peptides and proteins of fetal versus adult serum were analyzed by liquid chromatography, nano electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS). Three batches of fetal serum contained the Alpha Fetoprotein marker while adult serum batches did not. Insulin (INS), and insulin-like growth factor (ILGF), fibroblast growth factor (FGF), epidermal growth factor (EGF) and platelet derived growth factor (PDGF) were increased in fetal serum. New fetal growth factors including MEGF, HDGFRP and PSIP1 and soluble growth receptors such as TNFR, EGFR, NTRK2 and THRA were discovered. Addition of insulin or the homeotic transcription factor PSIP1, also referred to as Lens Epithelium Derived Growth Factor (LEDGF), partially restored the rounded phenotype of rapidly dividing cells but was not as effective as fetal serum. Thus, a new growth factor in fetal serum, LEDGF/PSIP1, was directly observed by tandem mass spectrometry and confirmed by add back experiments to cell culture media alongside insulin.

The plasma peptides of Alzheimer's disease.

BACKGROUND: A practical strategy to discover proteins specific to Alzheimer's dementia (AD) may be to compare the plasma peptides and proteins from patients with dementia to normal controls and patients with neurological conditions like multiple sclerosis or other diseases. The aim was a proof of principle for a method to discover proteins and/or peptides of plasma that show greater observation frequency and/or precursor intensity in AD. The endogenous tryptic peptides of Alzheimer's were compared to normals, multiple sclerosis, ovarian cancer, breast cancer, female normal, sepsis, ICU Control, heart attack, along with their institution-matched controls, and normal samples collected directly onto ice. METHODS: Endogenous tryptic peptides were extracted from blinded, individual AD and control EDTA plasma samples in a step gradient of acetonitrile for random and independent sampling by LC-ESI-MS/MS with a set of robust and sensitive linear quadrupole ion traps. The MS/MS spectra were fit to fully tryptic peptides within proteins identified using the X!TANDEM algorithm. Observation frequency of the identified proteins was counted using SEQUEST algorithm. The proteins with apparently increased observation frequency in AD versus AD Control were revealed graphically and subsequently tested by Chi Square analysis. The proteins specific to AD plasma by Chi Square with FDR correction were analyzed by the STRING algorithm. The average protein or peptide log10 precursor intensity was compared across disease and control treatments by ANOVA in the R statistical system. RESULTS: Peptides and/or phosphopeptides of common plasma proteins such as complement C2, C7, and C1QBP among others showed increased observation frequency by Chi Square and/or precursor intensity in AD. Cellular gene symbols with large Chi Square values (χ2 ≥ 25, p ≤ 0.001) from tryptic peptides included KIF12, DISC1, OR8B12, ZC3H12A, TNF, TBC1D8B, GALNT3, EME2, CD1B, BAG1, CPSF2, MMP15, DNAJC2, PHACTR4, OR8B3, GCK, EXOSC7, HMGA1 and NT5C3A among others. Similarly, increased frequency of tryptic phosphopeptides were observed from MOK, SMIM19, NXNL1, SLC24A2, Nbla10317, AHRR, C10orf90, MAEA, SRSF8, TBATA, TNIK, UBE2G1, PDE4C, PCGF2, KIR3DP1, TJP2, CPNE8, and NGF amongst others. STRING analysis showed an increase in cytoplasmic proteins and proteins associated with alternate splicing, exocytosis of luminal proteins, and proteins involved in the regulation of the cell cycle, mitochondrial functions or metabolism and apoptosis. Increases in mean precursor intensity of peptides from common plasma proteins such as DISC1, EXOSC5, UBE2G1, SMIM19, NXNL1, PANO, EIF4G1, KIR3DP1, MED25, MGRN1, OR8B3, MGC24039, POLR1A, SYTL4, RNF111, IREB2, ANKMY2, SGKL, SLC25A5, CHMP3 among others were associated with AD. Tryptic peptides from the highly conserved C-terminus of DISC1 within the sequence MPGGGPQGAPAAAGGGGVSHRAGSRDCLPPAACFR and ARQCGLDSR showed a higher frequency and highest intensity in AD compared to all other disease and controls. CONCLUSION: Proteins apparently expressed in the brain that were directly related to Alzheimer's including Nerve Growth Factor (NFG), Sphingomyelin Phosphodiesterase, Disrupted in Schizophrenia 1 (DISC1), the cell death regulator retinitis pigmentosa (NXNl1) that governs the loss of nerve cells in the retina and the cell death regulator ZC3H12A showed much higher observation frequency in AD plasma vs the matched control. There was a striking agreement between the proteins known to be mutated or dis-regulated in the brains of AD patients with the proteins observed in the plasma of AD patients from endogenous peptides including NBN, BAG1, NOX1, PDCD5, SGK3, UBE2G1, SMPD3 neuronal proteins associated with synapse function such as KSYTL4, VTI1B and brain specific proteins such as TBATA.

Re-evaluation of the rabbit myosin protein standard used to create the empirical statistical model for decoy library searching.

A Rabbit myosin standard, like that used to create the empirical statistical model, was randomly and independently sampled by liquid chromatography micro electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) with a linear quadrupole ion trap. The rabbit myosin protein standard appeared pure by SDS-PAGE and CBBR staining but showed many other proteins by silver staining. The LC-MS intensity from myosin and IgG samples were above the 99% safe limit of detection and quantification computed from 36 blank LC-ESI-MS/MS runs. The myosin contained ≤406 Gene Symbols, open reading frames or loci where 79 protein types showed ≥3 peptides from X!TANDEM. Myosins, actin, troponin, other proteins showed 95%-100% homology between the rabbit versus the human decoy library. The myosin protein complex from STRING was true positive compared to random or noise spectra MS/MS with a low type I error (p-value) and low FDR (q-value) computed in R. SDS-PAGE, Western blot, comparison to random and noise MS/MS spectra, X!TANDEM p-values, FDR corrected q-values, and STRING all agreed that the error rate of LC-ESI-MS/MS with a quadrupole ion trap is far below that assumed a priori by the design of the empirical statistical model for decoy library searching.

Capture and qualitative analysis of the activated Fc receptor complex from live cells.

This unit describes the isolation of activated Fc receptor complexes from RAW 264.7 macrophages using live-cell affinity receptor chromatography (LARC). The Fc receptor complex is activated and captured by IgG-coated microbeads on the surface of live macrophages. After the cells are disrupted, the receptor complexes are isolated by washing and sucrose gradient ultracentrifugation. Soluble proteins associated with the receptor complex are then eluted from the beads using a stepwise series of salt buffers and aqueous acetonitrile. The eluted proteins and the residual insoluble proteins on the beads can then be digested with trypsin and subjected to liquid chromatography, electrospray ionization, and tandem mass spectrometry (LC-ESI-MS/MS). Controls include IgG-coated beads incubated with crude cell lysates or growth medium and beads coated with oxidized LDL or bovine serum albumin. Using this method, proteins present in IgG-FcR complexes can be distinguished from those in control scavenger receptor complexes (oxLDL or BSA). Thus, LARC is capable of detecting specific members of IgG receptor supramolecular complexes.

Identification and quantification of peptides and proteins secreted from prostate epithelial cells by unbiased liquid chromatography tandem mass spectrometry using goodness of fit and analysis of variance.

The proteins secreted by prostate cancer cells (PC3(AR)6) were separated by strong anion exchange chromatography, digested with trypsin and analyzed by unbiased liquid chromatography tandem mass spectrometry with an ion trap. The spectra were matched to peptides within proteins using a goodness of fit algorithm that showed a low false positive rate. The parent ions for MS/MS were randomly and independently sampled from a log-normal population and therefore could be analyzed by ANOVA. Normal distribution analysis confirmed that the parent and fragment ion intensity distributions were sampled over 99.9% of their range that was above the background noise. Arranging the ion intensity data with the identified peptide and protein sequences in structured query language (SQL) permitted the quantification of ion intensity across treatments, proteins and peptides. The intensity of 101,905 fragment ions from 1421 peptide precursors of 583 peptides from 233 proteins separated over 11 sample treatments were computed together in one ANOVA model using the statistical analysis system (SAS) prior to Tukey-Kramer honestly significant difference (HSD) testing. Thus complex mixtures of proteins were identified and quantified with a high degree of confidence using an ion trap without isotopic labels, multivariate analysis or comparing chromatographic retention times.

Chi-square comparison of tryptic peptide-to-protein distributions of tandem mass spectrometry from blood with those of random expectation.

Proteomics uses tandem mass spectrometers and correlation algorithms to match peptides and their fragment spectra to amino acid sequences. The replication of multiple liquid chromatography experiments with electrospray ionization of peptides and tandem mass spectrometry (LC-ESI-MS/MS) produces large sets of MS/MS spectra. There is a need to assess the quality of large sets of experimental results by statistical comparison with that of random expectation. Classical frequency-based statistics such as goodness-of-fit tests for peptide-to-protein distributions could be used to calculate the probability that an entire set of experimental results has arisen by random chance. The frequency distributions of authentic MS/MS spectra from human blood were compared with those of false positive MS/MS spectra generated by a computer, or instrument noise, using the chi-square test. Here the mechanics of the chi-square test to compare the results in toto from a set of LC-ESI-MS/MS experiments with those of random expectation is detailed. The chi-square analysis of authentic spectra demonstrates unambiguously that the analysis of blood proteins separated by partition chromatography prior to tryptic digestions has a low probability that the cumulative peptide-to-protein distribution is the same as that of random or noise false positive spectra.

Peptide-to-protein distribution versus a competition for significance to estimate error rate in blood protein identification.

The simplest model-that authentic tandem mass spectrometry (MS/MS) spectra are no different from noise, random spectra, or false-positive results-may be directly examined by chi-square comparison of the peptide-to-protein distribution. The peptide-to-protein distribution of a set of 4151 redundant blood proteins identified by X!TANDEM indicated that there is a low probability that the authentic data were the same as noise, random spectra, or false-positive correlations (P<0.0001). In contrast, a competition for significance failed to distinguish approximately 90% of authentic blood proteins from those of noise, random spectra, or false-positive results (P<0.01) and apparently incurred a large type II error (false negative). The chi-square test of peptide-to-protein frequency distributions was found to be an efficient means to distinguish authentic data from false-positive results. Frequency-based statistics unambiguously demonstrated that proteins can be identified by liquid chromatography-electrospray ionization-MS/MS from human blood with acceptable confidence. Thus, the chi-square fit of the peptide-to-protein distribution could distinguish authentic data from random or false-positive data, but the score distribution method could not separate real results from false results.

Meta sequence analysis of human blood peptides and their parent proteins.

Sequence analysis of the blood peptides and their qualities will be key to understanding the mechanisms that contribute to error in LC-ESI-MS/MS. Analysis of peptides and their proteins at the level of sequences is much more direct and informative than the comparison of disparate accession numbers. A portable database of all blood peptide and protein sequences with descriptor fields and gene ontology terms might be useful for designing immunological or MRM assays from human blood. The results of twelve studies of human blood peptides and/or proteins identified by LC-MS/MS and correlated against a disparate array of genetic libraries were parsed and matched to proteins from the human ENSEMBL, SwissProt and RefSeq databases by SQL. The reported peptide and protein sequences were organized into an SQL database with full protein sequences and up to five unique peptides in order of prevalence along with the peptide count for each protein. Structured query language or BLAST was used to acquire descriptive information in current databases. Sampling error at the level of peptides is the largest source of disparity between groups. Chi Square analysis of peptide to protein distributions confirmed the significant agreement between groups on identified proteins.

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.

The endogenous peptides of human serum may have regulatory functions, have been associated with physiological states, and their modifications may reveal some mechanisms of disease. In order to correlate levels of specific peptides with disease alongside internal standards, the polypeptides must first be reliably extracted and identified. Endogenous blood peptides can be effectively enriched by precipitation of the serum with organic solvents followed by selective extraction of peptides using aqueous solutions modified with organic solvents. Polypeptides on filter paper were assayed with Coomasie brilliant blue binding. The polypeptides were resolved by detergent tricine polyacrylamide electrophoresis and visualized by diamine silver staining. Peptides in the extracts were collected by C18 and analyzed by matrix-assisted laser desorption/ionization and liquid chromatography-electrospray ionization-tandem mass spectrometry (MS/MS) quadrupole time-of-flight MS/MS. Peptides were resolved as multiple isotopic peaks in MS mode with mass deviation of 0.1 Da or less and similar accuracy for fragments. The sensitivity of MS and MS/MS analysis was estimated to be in the picomolar range or less. The peptide composition of the extracts was dependent on solvent formulation. Multiple peptides from apolipoproteins, complement proteins, coagulation factors, and many others were identified by X!Tandem with high mass accuracy of peptide ions and fragments from collision-induced dissociation. Many previously unreported posttranslational modifications of peptides including phosphorylations, oxidations, glycosylations, and others were detected with high mass accuracy and may be of clinical importance. About 4,630 redundant peptides were identified with 99% confidence separately, and together some 1,251 distinct proteins were identified with 99% confidence or greater using the Paragon algorithm.