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.

Mitochondria and cytochrome components released into the plasma of severe COVID-19 and ICU acute respiratory distress syndrome patients.

INTRODUCTION: Proteomic analysis of human plasma by LC-ESI-MS/MS has discovered a limited number of new cellular protein biomarkers that may be confirmed by independent biochemical methods. Analysis of COVID-19 plasma has indicated the re-purposing of known biomarkers that might be used as prognostic markers of COVID-19 infection. However, multiple molecular approaches have previously indicated that the SARS-COV2 infection cycle is linked to the biology of mitochondria and that the response to infections may involve the action of heme containing oxidative enzymes. METHODS: Human plasma from COVID-19 and ICU-ARDS was analyzed by classical analytical biochemistry techniques and classical frequency-based statistical approaches to look for prognostic markers of severe COVID-19 lung damage. Plasma proteins from COVID-19 and ICU-ARDS were identified and enumerated versus the controls of normal human plasma (NHP) by LC-ESI-MS/MS. The observation frequency of proteins detected in COVID-19 and ICU-ARDS patients were compared to normal human plasma, alongside random and noise MS/MS spectra controls, using the Chi Square (χ2) distribution. RESULTS: PCR showed the presence of MT-ND1 DNA in the plasma of COVID-19, ICU-ARDS, as well as normal human plasma. Mitochondrial proteins such as MRPL, L2HGDH, ATP, CYB, CYTB, CYP, NDUF and others, were increased in COVID-19 and ICU-ARDS plasma. The apparent activity of the cytochrome components were tested alongside NHP by dot blotting on PVDF against a purified cytochrome c standard preparation for H2O2 dependent reaction with luminol as measured by enhanced chemiluminescence (ECL) that showed increased activity in COVID-19 and ICU-ARDS patients. DISCUSSION: The results from PCR, LC-ESI-MS/MS of tryptic peptides, and cytochrome ECL assays confirmed that mitochondrial components were present in the plasma, in agreement with the established central role of the mitochondria in SARS-COV-2 biology. The cytochrome activity assay showed that there was the equivalent of at least nanogram amounts of cytochrome(s) in the plasma sample that should be clearly detectable by LC-ESI-MS/MS. The release of the luminol oxidase activity from cells into plasma forms the basis of a simple and rapid test for the severity of cell damage and lung injury in COVID-19 infection and ICU-ARDS.

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.

Linear and Gaussian Analysis of a Single Enzyme Molecule by LC-MS.

The alkaline phosphatase-streptavidin enzyme amplification conjugate (APSA) was diluted and quantified to the equivalent of one enzyme molecule injected on column by monitoring the production of excess adenosine from adenosine monophosphate (AMP) using sensitive and selective enzyme-linked mass spectrometric assay. The APSA enzyme conjugate has a mass of about 195 kDa and catalyzed the production of millions of enzyme products over the course of incubation that may be sensitively quantified by liquid chromatography, electrospray ionization, and mass spectrometry. APSA enzyme conjugate from fg/mL to ag/mL alongside 0 g/mL (control) was incubated with the substrate 1 mM AMP for 2 h in free solution before collecting a 1 µL of sample of the enzyme product adenosine for injection and analysis by LC-MS. The enzyme product adenosine showed a Gaussian distribution after log10 transformation. The safe limit of detection and quantification was approximately 250 zg of APSA enzyme conjugate injected on column. A linear signal with acceptable error was observed at the mass of the enzyme product adenosine from 10 to 10000 zg of APSA enzyme conjugate injected, compared to controls without enzyme. It was possible to make a linear and Gaussian measurement to the single molecule range of the universal APSA enzyme amplification conjugate per micro liter injected with approximately 10% error. This study describes the first linear and Gaussian quantification of enzyme product from the equivalent of one enzyme conjugate molecule injected onto LC-MS for analysis.

The plasma peptides of sepsis.

BACKGROUND: A practical strategy to discover sepsis specific proteins may be to compare the plasma peptides and proteins from patients in the intensive care unit with and without sepsis. The aim was to discover proteins and/or peptides that show greater observation frequency and/or precursor intensity in sepsis. The endogenous tryptic peptides of ICU-Sepsis were compared to ICU Control, ovarian cancer, breast cancer, female normal, sepsis, heart attack, Alzheimer's and multiple sclerosis along with their institution-matched controls, female normals and normal samples collected directly onto ice. METHODS: Endogenous tryptic peptides were extracted from individual sepsis 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 using the X!TANDEM algorithm. The protein observation frequency was counted using the SEQUEST algorithm after selecting the single best charge state and peptide sequence for each MS/MS spectra. The protein observation frequency of ICU-sepsis versus ICU Control was subsequently tested by Chi square analysis. 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 ITIH3, SAA2, SAA1, and FN1 showed increased observation frequency by Chi square (χ2 > 9, p < 0.003) and/or precursor intensity in sepsis. Cellular gene symbols with large Chi square values from tryptic peptides included POTEB, CTNNA1, U2SURP, KIF24, NLGN2, KSR1, GTF2H1, KIT, RPS6KL1, VAV2, HSPA7, SMC2, TCEB3B, ZNF300, SUPV3L1, ADAMTS20, LAMB4, MCCC1, SUPT6H, SCN9A, SBNO1, EPHA1, ABLIM2, cB5E3.2, EPHA10, GRIN2B, HIVEP2, CCL16, TKT, LRP2 and TMF1 amongst others showed increased observation frequency. Similarly, increased frequency of tryptic phosphopeptides were observed from POM121C, SCN8A, TMED8, NSUN7, SLX4, MADD, DNLZ, PDE3B, UTY, DEPDC7, MTX1, MYO1E, RXRB, SYDE1, FN1, PUS7L, FYCO1, USP26, ACAP2, AHI1, KSR2, LMAN1, ZNF280D and SLC8A2 amongst others. Increases in mean precursor intensity in peptides from common plasma proteins such as ITIH3, SAA2, SAA1, and FN1 as well as cellular proteins such as COL24A1, POTEB, KANK1, SDCBP2, DNAH11, ADAMTS7, MLLT1, TTC21A, TSHR, SLX4, MTCH1, and PUS7L among others were associated with sepsis. The processing of SAA1 included the cleavage of the terminal peptide D/PNHFRPAGLPEKY from the most hydrophilic point of SAA1 on the COOH side of the cystatin C binding that was most apparent in ICU-Sepsis patients compared to all other diseases and controls. Additional cleavage of SAA1 on the NH2 terminus side of the cystatin binding site were observed in ICU-Sepsis. Thus there was disease associated variation in the processing of SAA1 in ICU-Sepsis versus ICU controls or other diseases and controls. CONCLUSION: Specific proteins and peptides that vary between diseases might be discovered by the random and independent sampling of multiple disease and control plasma from different hospital and clinics by LC-ESI-MS/MS for storage in a relational SQL Server database and analysis with the R statistical system that will be a powerful tool for clinical research. The processing of SAA1 may play an unappreciated role in the inflammatory response to Sepsis.

Re-evaluation of the 18 non-human protein standards used to create the empirical statistical model for decoy library searching.

The Empirical Statistical Model (ESM) for decoy library searching fused the expected amino acid sequence of 18 non-human protein standards to a human decoy library. The ESM assumed a priori the standards were pure such that only the 18 nominal proteins were true positive, all other proteins were false positive, there was no overlap in the peptides of non-human proteins versus human proteins, and that the score distribution of individual peptides would resolve true positive from false positive results or noise. The results of random and independent sampling by LC-ESI-MS/MS indicated that the fundamental assumptions of the ESM were not in good agreement with the actual purity of the commercial test standards and so the method showed a 99.7% false negative rate. The ESM for decoy library searching apparently showed poor agreement with SDS-PAGE using silver staining, goodness of fit of MS/MS spectra by X!TANDEM, FDR correction by Benjamini and Hochberg, or comparison to the observation frequency of null random MS/MS spectra, that all confirmed the standards contain hundreds of proteins with a low FDR of primary structural identification. The protein observation frequency increased with abundance and the log10 precursor intensity distributions were Gaussian and nearly ideal for relative quantification.

The plasma peptides of breast versus ovarian cancer.

BACKGROUND: There is a need to demonstrate a proof of principle that proteomics has the capacity to analyze plasma from breast cancer versus other diseases and controls in a multisite clinical trial design. The peptides or proteins that show a high observation frequency, and/or precursor intensity, specific to breast cancer plasma might be discovered by comparison to other diseases and matched controls. The endogenous tryptic peptides of breast cancer plasma were compared to ovarian cancer, female normal, sepsis, heart attack, Alzheimer's and multiple sclerosis along with the institution-matched normal and control samples collected directly onto ice. METHODS: Endogenous tryptic peptides were extracted from individual breast cancer and control EDTA plasma samples in a step gradient of acetonitrile, and collected over preparative C18 for LC-ESI-MS/MS with a set of LTQ XL linear quadrupole ion traps working together in parallel to randomly and independently sample clinical populations. The MS/MS spectra were fit to fully tryptic peptides or phosphopeptides within proteins using the X!TANDEM algorithm. The protein observation frequency was counted using the SEQUEST algorithm after selecting the single best charge state and peptide sequence for each MS/MS spectra. The observation frequency was subsequently tested by Chi Square analysis. The log10 precursor intensity was compared by ANOVA in the R statistical system. RESULTS: Peptides and/or phosphopeptides of common plasma proteins such as APOE, C4A, C4B, C3, APOA1, APOC2, APOC4, ITIH3 and ITIH4 showed increased observation frequency and/or precursor intensity in breast cancer. Many cellular proteins also showed large changes in frequency by Chi Square (χ2 > 100, p < 0.0001) in the breast cancer samples such as CPEB1, LTBP4, HIF-1A, IGHE, RAB44, NEFM, C19orf82, SLC35B1, 1D12A, C8orf34, HIF1A, OCLN, EYA1, HLA-DRB1, LARS, PTPDC1, WWC1, ZNF562, PTMA, MGAT1, NDUFA1, NOGOC, OR1E1, OR1E2, CFI, HSA12, GCSH, ELTD1, TBX15, NR2C2, FLJ00045, PDLIM1, GALNT9, ASH2L, PPFIBP1, LRRC4B, SLCO3A1, BHMT2, CS, FAM188B2, LGALS7, SAT2, SFRS8, SLC22A12, WNT9B, SLC2A4, ZNF101, WT1, CCDC47, ERLIN1, SPFH1, EID2, THOC1, DDX47, MREG, PTPRE, EMILIN1, DKFZp779G1236 and MAP3K8 among others. The protein gene symbols with large Chi Square values were significantly enriched in proteins that showed a complex set of previously established functional and structural relationships by STRING analysis. An increase in mean precursor intensity of peptides was observed for QSER1 as well as SLC35B1, IQCJ-SCHIP1, MREG, BHMT2, LGALS7, THOC1, ANXA4, DHDDS, SAT2, PTMA and FYCO1 among others. In contrast, the QSER1 peptide QPKVKAEPPPK was apparently specific to ovarian cancer. CONCLUSION: There was striking agreement between the breast cancer plasma peptides and proteins discovered by LC-ESI-MS/MS with previous biomarkers from tumors, cells lines or body fluids by genetic or biochemical methods. The results indicate that variation in plasma peptides from breast cancer versus ovarian cancer may be directly discovered by LC-ESI-MS/MS that will be a powerful tool for clinical research. It may be possible to use a battery of sensitive and robust linear quadrupole ion traps for random and independent sampling of plasma from a multisite clinical trial.

The plasma peptidome.

BACKGROUND: It may be possible to discover new diagnostic or therapeutic peptides or proteins from blood plasma using LC-ESI-MS/MS to identify, with a linear quadrupole ion trap to identify, quantify and compare the statistical distributions of peptides cleaved ex vivo from plasma samples from different clinical populations. METHODS: A systematic method for the organic fractionation of plasma peptides was applied to identify and quantify the endogenous tryptic peptides from human plasma from multiple institutions by C18 HPLC followed nano electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) with a linear quadrupole ion trap. The endogenous tryptic peptides, or tryptic phospho peptides (i.e. without exogenous digestion), were extracted in a mixture of organic solvent and water, dried and collected by preparative C18. The tryptic peptides from 6 institutions with 12 different disease and normal EDTA plasma populations, alongside ice cold controls for pre-analytical variation, were characterized by mass spectrometry. Each patient plasma was precipitated in 90% acetonitrile and the endogenous tryptic peptides extracted by a stepwise gradient of increasing water and then formic acid resulting in 10 sub-fractions. The fractionated peptides were manually collected over preparative C18 and injected for 1508 LC-ESI-MS/MS experiments analyzed in SQL Server R. RESULTS: Peptides that were cleaved in human plasma by a tryptic activity ex vivo provided convenient and sensitive access to most human proteins in plasma that show differences in the frequency or intensity of proteins observed across populations that may have clinical significance. Combination of step wise organic extraction of 200 µL of plasma with nano electrospray resulted in the confident identification and quantification ~ 14,000 gene symbols by X!TANDEM that is the largest number of blood proteins identified to date and shows that you can monitor the ex vivo proteolysis of most human proteins, including interleukins, from blood. A total of 15,968,550 MS/MS spectra ≥ E4 intensity counts were correlated by the SEQUEST and X!TANDEM algorithms to a federated library of 157,478 protein sequences that were filtered for best charge state (2+ or 3+) and peptide sequence in SQL Server resulting in 1,916,672 distinct best-fit peptide correlations for analysis with the R statistical system. SEQUEST identified some 140,054 protein accessions, or some ~ 26,000 gene symbols, proteins or loci, with at least 5 independent correlations. The X!TANDEM algorithm made at least 5 best fit correlations to more than 14,000 protein gene symbols with p-values and FDR corrected q-values of ~ 0.001 or less. Log10 peptide intensity values showed a Gaussian distribution from E8 to E4 arbitrary counts by quantile plot, and significant variation in average precursor intensity across the disease and controls treatments by ANOVA with means compared by the Tukey-Kramer test. STRING analysis of the top 2000 gene symbols showed a tight association of cellular proteins that were apparently present in the plasma as protein complexes with related cellular components, molecular functions and biological processes. CONCLUSIONS: The random and independent sampling of pre-fractionated blood peptides by LC-ESI-MS/MS with SQL Server-R analysis revealed the largest plasma proteome to date and was a practical method to quantify and compare the frequency or log10 intensity of individual proteins cleaved ex vivo across populations of plasma samples from multiple clinical locations to discover treatment-specific variation using classical statistics suitable for clinical science. It was possible to identify and quantify nearly all human proteins from EDTA plasma and compare the results of thousands of LC-ESI-MS/MS experiments from multiple clinical populations using standard database methods in SQL Server and classical statistical strategies in the R data analysis system.

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.

A method for the extraction of the endogenous tryptic peptides (peptidome) from human EDTA plasma.

The proteins identified from endogenous peptides agreed between serum versus plasma, and tryptic versus non-tryptic peptides, when collected by C18 alone and analyzed by liquid chromatography electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) including amyloids, apolipoproteins, haptoglobin, complements, fibrinogens, hemopexin, antitrypsin and alpha 2 macroglobulin. Precipitation of polypeptides from plasma in 9 vol of 100% organic solvent followed by stepwise extraction of the insoluble pellet with an increasing fraction of water identified thousands of proteins. A Coomassie-blue protein binding assay, and tricine SDS-PAGE, showed that Acetonitrile-Water (AH) resulted in a greater relative enrichment of low molecular weight plasma polypeptides than Acetonitrile-Methanol Water (AMH). A total of 905,386 MS/MS spectra greater than ~10,000 (E4) counts were correlated by X!TANDEM to a federated human protein library of 153,124 different protein sequences that resulted in 58,223 fully tryptic peptides from 3463 Gene Symbols of which 1880 had ≥ 5 independent peptides (p ≤ 0.00001). The results were filtered and organized in an SQL database for analysis using the generic R statistical analysis system. Cellular proteins including secreted and exosome proteins, signaling factors, nucleic acid binding proteins, metabolic enzymes and uncharacterized factors were observed with a significant enrichment of expected protein-protein interactions by STRING analysis.

The plasma peptides of ovarian cancer.

BACKGROUND: It may be possible to discover new diagnostic or therapeutic peptides or proteins from blood plasma by using liquid chromatography and tandem mass spectrometry to identify, quantify and compare the peptides cleaved ex vivo from different clinical populations. The endogenous tryptic peptides of ovarian cancer plasma were compared to breast cancer and female cancer normal controls, other diseases with their matched or normal controls, plus ice cold plasma to control for pre-analytical variation. METHODS: The endogenous tryptic peptides or tryptic phospho peptides (i.e. without exogenous digestion) were analyzed from 200 µl of EDTA plasma. The plasma peptides were extracted by a step gradient of organic/water with differential centrifugation, dried, and collected over C18 for analytical HPLC nano electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) with a linear quadrupole ion trap. The endogenous peptides of ovarian cancer were compared to multiple disease and normal samples from different institutions alongside ice cold controls. Peptides were randomly and independently sampled by LC-ESI-MS/MS. Precursor ions from peptides > E4 counts were identified by the SEQUEST and X!TANDEM algorithms, filtered in SQL Server, before testing of frequency counts by Chi Square (χ2), for analysis with the STRING algorithm, and comparison of precursor intensity by ANOVA in the R statistical system with the Tukey-Kramer Honestly Significant Difference (HSD) test. RESULTS: Peptides and/or phosphopeptides of common plasma proteins such as HPR, HP, HPX, and SERPINA1 showed increased observation frequency and/or precursor intensity in ovarian cancer. Many cellular proteins showed large changes in frequency by Chi Square (χ2 > 60, p < 0.0001) in the ovarian cancer samples such as ZNF91, ZNF254, F13A1, LOC102723511, ZNF253, QSER1, P4HA1, GPC6, LMNB2, PYGB, NBR1, CCNI2, LOC101930455, TRPM5, IGSF1, ITGB1, CHD6, SIRT1, NEFM, SKOR2, SUPT20HL1, PLCE1, CCDC148, CPSF3, MORN3, NMI, XTP11, LOC101927572, SMC5, SEMA6B, LOXL3, SEZ6L2, and DHCR24. The protein gene symbols with large Chi Square values were significantly enriched in proteins that showed a complex set of previously established functional and structural relationships by STRING analysis. Analysis of the frequently observed proteins by ANOVA confirmed increases in mean precursor intensity in ZFN91, TRPM5, SIRT1, CHD6, RIMS1, LOC101930455 (XP_005275896), CCDC37 and GIMAP4 between ovarian cancer versus normal female and other diseases or controls by the Tukey-Kramer HSD test. CONCLUSION: Here we show that separation of endogenous peptides with a step gradient of organic/water and differential centrifugation followed by random and independent sampling by LC-ESI-MS/MS with analysis of peptide frequency and intensity by SQL Server and R revealed significant difference in the ex vivo cleavage of peptides between ovarian cancer and other clinical treatments. There was striking agreement between the proteins discovered from cancer plasma versus previous biomarkers discovered in tumors by genetic or biochemical methods. The results indicate that variation in plasma proteins from ovarian cancer may be directly discovered by LC-ESI-MS/MS that will be a powerful tool for clinical research.

OxLDL receptor chromatography from live human U937 cells identifies SYK(L) that regulates phagocytosis of oxLDL.

The binding and activation of macrophages by microscopic aggregates of oxLDL in the intima of the arteries may be an important step towards atherosclerosis leading to heart attack and stroke. Microbeads coated with oxLDL were used to activate, capture and isolate the oxLDL receptor complex from the surface of live cells. Analysis of the resulting tryptic peptides by liquid chromatography and tandem mass spectrometry revealed the Spleen Tyrosine Kinase (SYK), and many of SYK's known interaction network including Fc receptors (FCGR2A, FCER1G and FCGR1A) Toll receptor 4 (TLR4), receptor kinases like EGFRs, as well as RNA binding and metabolism proteins. High-intensity precursor ions (∼9*E3 to 2*E5 counts) were correlated to peptides and specific phosphopeptides from long isoform of SYK (SYK-L) by the SEQUEST, OMSSA and X!TANDEM algorithms. Peptides or phosphopeptides from SYK were observed with the oxLDL-microbeads. Pharmacological inhibitors of SYK activity significantly reduced the engulfment of oxLDL microbeads in the presence of serum factors, but had little effect on IgG phagocytosis. Anti SYK siRNA regulated oxLD engulfment in the context of serum factors and or SYK-L siRNA significantly inhibited engulfment of oxLDL microbeads, but not IgG microbeads.

Linear quantification of a streptavidin-alkaline phosphatase probe for enzyme-linked immuno mass spectrometric assay.

The alkaline phosphatase-streptavidin (AP-SA) probe released adenosine (∼267.2 Da) from the substrate adenosine monophosphate (AMP), where a signal may be detected from as little as 0.5 µl of a 0.1-pg/ml dilution of the probe (2.6 × 10(-22) mol). The signal from the AP-SA probe was linear from 1 to 50 pg/ml by monitoring adenosine release at 268 m/z (M + H) with liquid chromatography, electrospray ionization, and quadrupole mass spectrometry (LC-ESI-MS). The safe limit of detection and quantification of the AP-SA probe was approximately 0.5 pg/well or 5 pg/ml. Enzyme-linked immuno mass spectrometric assay (ELIMSA) using the AP-SA probe provided a linear signal response for prostate-specific antigen (PSA) against external standards from 1 to 500 pg/ml. The ELIMSA showed a safe limit of detection and quantification at 5 pg PSA/well or 50 pg/ml (false positive detection rate P ≤ 0.01). Female samples of 100 µl plasma/well were read against standards and blanks made in normal female plasma, and the lowest sample quantified was approximately 9.8 pg/well or 98 pg/ml. Here ELIMSA was applied to measure PSA in plasma from female, normal male, prostatectomy patient, and cancer patient samples that showed significant differences by analysis of variance (ANOVA).

A phagocytosis assay for oxidized low-density lipoprotein versus immunoglobulin G-coated microbeads in human U937 macrophages.

The human monocyte cell line U937 was differentiated into an adherent macrophage phenotype using phorbol 12-myristate 13-acetate (PMA) to assay the phagocytosis of oxidized low-density lipoprotein (oxLDL) that may play a role in atherosclerosis. Microbeads were coated with the inflammatory ligand oxLDL to create a novel phagocytosis assay that models the binding of macrophages to oxLDL in the solid phase such as found in the fatty streaks of the arteries. The oxLDL was prepared with LDL from human ethylenediaminetetraacetic acid (EDTA) plasma oxidized with an excess (5 mM) of the strong oxidizing agent CuSO4 and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Western blot. The binding of the oxLDL to the beads was confirmed by DilC18-oxLDL staining and confocal microscopy in addition to trypsin digestion of the microbeads for liquid chromatography, electrospray ionization, and tandem mass spectrometry. Phagocytosis of the oxLDL versus human bulk immunoglobulin G1 (IgG1)-coated microbeads was assayed over time, in the presence and absence of serum factors, by pulse chase and with enzyme inhibitor treatments. The ligand beads were then stained with specific antibodies to oxLDL versus human IgG to differentially stain external versus engulfed ligand microbeads. The phagocytosis of oxLDL and IgG ligand microbeads was abolished by the actin polymerization inhibitors cytochalasin D and latrunculin. Pharmacological inhibitors of the receptor enzymes JAK, SRC, and PLC prevented both IgG and oxLDL receptor function. In contrast, the function of the oxLDL phagocytic receptor complex was more sensitive to inhibition of PTK2, PKC, and SYK activity.

AMP-Activated Protein Kinase Regulates the Cell Surface Proteome and Integrin Membrane Traffic.

The cell surface proteome controls numerous cellular functions including cell migration and adhesion, intercellular communication and nutrient uptake. Cell surface proteins are controlled by acute changes in protein abundance at the plasma membrane through regulation of endocytosis and recycling (endomembrane traffic). Many cellular signals regulate endomembrane traffic, including metabolic signaling; however, the extent to which the cell surface proteome is controlled by acute regulation of endomembrane traffic under various conditions remains incompletely understood. AMP-activated protein kinase (AMPK) is a key metabolic sensor that is activated upon reduced cellular energy availability. AMPK activation alters the endomembrane traffic of a few specific proteins, as part of an adaptive response to increase energy intake and reduce energy expenditure. How increased AMPK activity during energy stress may globally regulate the cell surface proteome is not well understood. To study how AMPK may regulate the cell surface proteome, we used cell-impermeable biotinylation to selectively purify cell surface proteins under various conditions. Using ESI-MS/MS, we found that acute (90 min) treatment with the AMPK activator A-769662 elicits broad control of the cell surface abundance of diverse proteins. In particular, A-769662 treatment depleted from the cell surface proteins with functions in cell migration and adhesion. To complement our mass spectrometry results, we used other methods to show that A-769662 treatment results in impaired cell migration. Further, A-769662 treatment reduced the cell surface abundance of ß1-integrin, a key cell migration protein, and AMPK gene silencing prevented this effect. While the control of the cell surface abundance of various proteins by A-769662 treatment was broad, it was also selective, as this treatment did not change the cell surface abundance of the transferrin receptor. Hence, the cell surface proteome is subject to acute regulation by treatment with A-769662, at least some of which is mediated by the metabolic sensor AMPK.

An enzyme-linked immuno-mass spectrometric assay with the substrate adenosine monophosphate.

An enzyme-linked immuno-mass spectrometric assay (ELIMSA) with the specific detection probe streptavidin conjugated to alkaline phosphatase catalyzed the production of adenosine from the substrate adenosine monophosphate (AMP) for sensitive quantification of prostate-specific antigen (PSA) by mass spectrometry. Adenosine ionized efficiently and was measured to the femtomole range by dilution and direct analysis with micro-liquid chromatography, electrospray ionization, and mass spectrometry (LC-ESI-MS). The LC-ESI-MS assay for adenosine production was shown to be linear and accurate using internal (13)C(15)N adenosine isotope dilution, internal (13)C(15)N adenosine one-point calibration, and external adenosine standard curves with close agreement. The detection limits of LC-ESI-MS for alkaline phosphatase-streptavidin (AP-SA, ∼190,000 Da) was tested by injecting 0.1 µl of a 1 pg/ml solution, i.e., 100 attograms or 526 yoctomole (5.26E-22) of the alkaline-phosphatase labeled probe on column (about 315 AP-SA molecules). The ELIMSA for PSA was linear and showed strong signals across the picogram per milliliter range and could robustly detect PSA from all of the prostatectomy patients and all of the female plasma samples that ranged as low as 70 pg/ml with strong signals well separated from the background and well within the limit of quantification of the AP-SA probe. The results of the ELIMSA assay for PSA are normal and homogenous when independently replicated with a fresh standard over multiple days, and intra and inter diem assay variation was less than 10 % of the mean. In a blind comparison, ELIMSA showed excellent agreement with, but was more sensitive than, the present gold standard commercial fluorescent ELISA, or ECL-based detection, of PSA from normal and prostatectomy samples, respectively.

Pyridoxamine-5-phosphate enzyme-linked immune mass spectrometric assay substrate for linear absolute quantification of alkaline phosphatase to the yoctomole range applied to prostate specific antigen.

There is a need to measure proteins that are present in concentrations below the detection limits of existing colorimetric approaches with enzyme-linked immunoabsorbent assays (ELISA). The powerful enzyme alkaline phosphatase conjugated to the highly specific bacterial protein streptavidin binds to biotinylated macromolecules like proteins, antibodies, or other ligands and receptors with a high affinity. The binding of the biotinylated detection antibody, with resulting amplification of the signal by the catalytic production of reporter molecules, is key to the sensitivity of ELISA. The specificity and amplification of the signal by the enzyme alkaline phosphatase in ELISA together with the sensitivity of liquid chromatography electrospray ionization and mass spectrometry (LC-ESI-MS) to detect femtomole to picomole amounts of reporter molecules results in an ultrasensitive enzyme-linked immune mass spectrometric assay (ELIMSA). The novel ELIMSA substrate pyridoxamine-5-phosphate (PA5P) is cleaved by the enzyme alkaline phosphatase to yield the basic and hydrophilic product pyridoxamine (PA) that elutes rapidly with symmetrical peaks and a flat baseline. Pyridoxamine (PA) and (13)C PA were both observed to show a linear relationship between log ion intensity and quantity from picomole to femtomole amounts by liquid chromatography-electrospray ionization and mass spectrometry. Four independent methods, (i) internal (13)C isotope PA dilution curves, (ii) internal (13)C isotope one-point calibration, (iii) external PA standard curve, and (iv) external (13)C PA standard curve, all agreed within 1 digit in the same order of magnitude on the linear quantification of PA. Hence, a mass spectrometer can be used to robustly detect 526 ymol of the alkaline phosphatase streptavidin probe and accurately quantify zeptomole amounts of PSA against log linear absolute standard by micro electrospray on a simple ion trap.

Enzyme linked immuno mass spectrometric assay (ELIMSA).

A new technology termed ELIMSA combines the specificity and enzymatic amplification of Enzyme Linked Immunosorbent assay (ELISA) with the sensitivity and flexibility of mass spectrometry (MS). At present, substrates for the reporter enzymes horseradish peroxidase (HRP) or alkaline phosphatase (AP) yield colored, fluorescent or luminescent products. The central concept of ELIMSA is that the reporter enzymes HRP and AP yield products that ionize efficiently with a high signal to noise ratio that can be measured by mass spectrometry. The reporter enzymes HRP or AP may be covalently attached to a specific detection probe such as a protein or an antibody to bind their target analyte and then catalyze the rapid production of ionizable, small-molecules. The use of mass spectrometry to measure small molecule products may commonly reach femto to picomol amounts on the column with high signal to noise ratio. Mass spectrometry combined with the enzyme amplification in ELISA provides absolute sensitivity to detect attomol of PSA and was comparable to, or more sensitive, than radio immune assays and electrochemical detectors but with only existing reagents and equipment. ELIMSA permits monitoring of multiple substrates and products and provides comparison to absolute standards. BIOLOGICAL SIGNIFICANCE: There is an urgent need to detect and quantify low abundance proteins such as hormones, chemokines, cytokines, and others that exist at attomolar concentrations under physiological conditions by ELIMSA. A sensitive method for the quantification of immunological assays is obtained by applying mass spectrometry to detect the products of the alkaline phosphatase (AP) and horseradish peroxidase (HRP) enzyme reactions. There are many molecules from human subjects or micro-organisms that are of great importance to medicine, industry, nutrition or the environment that need to be repeatedly analyzed and are often near to, or beyond, the edge of existing analytical technology. The presence of molecules in biological samples, industrial products or the environment may be detected by probes that bind to the target analyte. Combining the reporter enzymes from ELISA with sensitive liquid chromatography (LC), electrospray ionization (ESI) and tandem mass spectrometry (MS/MS) will permit the sensitive detection and quantification of the molecular probes by Enzyme Linked Immuno Mass Spectrometric Assay (ELIMSA). The flexibility and sensitivity of mass spectrometry to measure large numbers of compounds simultaneously should permit the quantification of multiple ELIMSA reactions at separate mass-to-charge (m/z) ratios. Hence ELIMSA and it variants should permit the rapid and simple detection and quantification of many molecules over the complete range of biologically important concentrations without the use of radiolabels using only existing antibodies, reagents and instruments. Antibodies coupled to reporter enzymes that are widely used in biomedical and environmental applications can now be detected and quantified using ultra sensitive mass spectrometry to create a sensitive and flexible ELIMSA system. Absolute standards of analytes or enzyme product may serve as a reference.