Application of Targeted Quantitative Proteomics Analysis in Human

Here, we report the implementation and application of a LC MALDI TOF/TOF-based platform for targeted quantitative proteomics analysis in human CSF...
0 downloads 0 Views 1MB Size
Application of Targeted Quantitative Proteomics Analysis in Human Cerebrospinal Fluid Using a Liquid Chromatography Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Tandem Mass Spectrometer (LC MALDI TOF/TOF) Platform Sheng Pan,*,† John Rush,‡ Elaine R. Peskind,§ Douglas Galasko,| Kathryn Chung,⊥ Joseph Quinn,⊥ Joseph Jankovic,# James B. Leverenz,§,¶,£ Cyrus Zabetian,¶,£,∞ Catherine Pan,† Yan Wang,† Jung Hun Oh,@ Jean Gao,@ Jianpeng Zhang,† Thomas Montine,† and Jing Zhang*,† Department of Pathology, University of Washington, Seattle, Washington 98195, Cell Signaling Technology, Inc., Danvers, Massachusetts 01915, Department of Psychiatry and Behavioral Sciences, University of Washington School of Medicine, Seattle, Washington 98195, Department of Neurosciences, University of California, San Diego, California 92093, Department of Neurology, Oregon Health and Science University, Portland, Oregon 97239, Department of Neurology, Baylor College of Medicine, Houston, Texas 77030, Department of Neurology, University of Washington School of Medicine, Seattle, Washington 98195, VA Northwest Network Mental Illness Research, Education, and Clinical Center, VA Puget Sound Health Care System, Seattle, Washington 98108, Geriatric Research, Education, and Clinical Center, VA Puget Sound Health Care System, Seattle, Washington 98108, and Department of Computer Science and Engineering, University of Texas, Arlington, Texas 76019 Received October 1, 2007

Targeted quantitative proteomics by mass spectrometry aims to selectively detect one or a panel of peptides/proteins in a complex sample and is particularly appealing for novel biomarker verification/validation because it does not require specific antibodies. Here, we demonstrated the application of targeted quantitative proteomics in searching, identifying, and quantifying selected peptides in human cerebrospinal spinal fluid (CSF) using a matrix-assisted laser desorption/ ionization time-of-flight tandem mass spectrometer (MALDI TOF/TOF)-based platform. The approach involved two major components: the use of isotopic-labeled synthetic peptides as references for targeted identification and quantification and a highly selective mass spectrometric analysis based on the unique characteristics of the MALDI instrument. The platform provides high confidence for targeted peptide detection in a complex system and can potentially be developed into a high-throughput system. Using the liquid chromatography (LC) MALDI TOF/TOF platform and the complementary identification strategy, we were able to selectively identify and quantify a panel of targeted peptides in the whole proteome of CSF without prior depletion of abundant proteins. The effectiveness and robustness of the approach associated with different sample complexity, sample preparation strategies, as well as mass spectrometric quantification were evaluated. Other issues related to chromatography separation and the feasibility for high-throughput analysis were also discussed. Finally, we applied targeted quantitative proteomics to analyze a subset of previously identified candidate markers in CSF samples of patients with Parkinson’s disease (PD) at different stages and Alzheimer’s disease (AD) along with normal controls. Keywords: Mass spectrometry • quantitative proteomics • matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (MALDI TOF/TOF) • cerebrospinal fluid • biomarker • aging • Parkinson’s disease • Alzheimer’s disease

Introduction Discovery-based proteomics has greatly enhanced our ability to reveal candidate proteins involved in various human diseases or biological settings. However, it is extremely challenging to * To whom correspondence should be addressed: Department of Pathology, University of Washington School of Medicine, Seattle, Washington, WA. E-mail: [email protected] (J.Z.); [email protected] (S.P.). † University of Washington. ‡ Cell Signaling Technology, Inc. § Department of Psychiatry and Behavioral Sciences, University of Washington School of Medicine. | University of California. ⊥ Oregon Health and Science University. # Baylor College of Medicine. ¶ Department of Neurology, University of Washington School of Medicine. £ VA Northwest Network Mental Illness Research, Education, and Clinical Center, VA Puget Sound Health Care System. ∞ Geriatric Research, Education, and Clinical Center, VA Puget Sound Health Care System. @ University of Texas.

720 Journal of Proteome Research 2008, 7, 720–730 Published on Web 01/11/2008

validate these putative markers largely because of the enormous complexity of biological systems, heterogeneity of humans, and/or lack of high-throughput technology.1,2 At the protein level, the most widely used technology to date for biomarker verification and validation is the enzyme-linked immunosorbent assay (ELISA) or multiplex Luminex platform (MAP).3,4 The difficulty with the ELISA/MAP approach centers on the availability of specific antibodies against novel proteins identified by proteomics. For instance, in our recent study, where multiple protein marker candidates were revealed to be uniquely associated with Alzheimer’s disease (AD) and Parkinson’s disease (PD) using global profiling, a majority of novel and brain-specific proteins could not be verified/validated because of the lack of commercially available specific antibodies.5 To circumvent this limitation, proteomics technologies targeted at unique proteins/peptides within a complex background are being actively developed.6–9 10.1021/pr700630x CCC: $40.75

 2008 American Chemical Society

Analysis of Human Cerebrospinal Fluid Using LC MALDI TOF/TOF Mass-spectrometry-based targeted proteomics for biomarker verification/validation is comparable to modern advanced antibody-based approaches; in that, it offers multiplexing detection capability with an excellent dynamic range. In addition, because peptide identification can be achieved precisely via high mass accuracy and/or peptide sequencing using collision-induced dissociation (CID), the mass spectrometric approaches provide high specificity for peptide/protein identification, which is critical to biomarker verification/ validation in complex systems. To perform peptide-based quantitative targeted proteomics, most investigators have been focusing on an electrospray ionization (ESI)-based triplequadrupole mass spectrometer using a multiple reaction monitoring (MRM) technique,10,11 which has been used for decades in the pharmaceutical industry for detection of small molecules. Indeed, the maturity and the highly developed automation of this technology have provided great convenience in adapting the methodology for targeted quantitative proteomics. More recently, matrix-assisted laser desorption/ionization (MALDI)-based instruments 12,13 have been increasingly used in proteomics because of the advances in instrumentation and application platforms, including the coupling of offline liquid chromatography (LC)/spotting for sample separation prior to mass spectrometric analysis and the introduction of tandem instruments to allow for fragmentation and sequencing of peptides for identification. In comparison to the ESI-based instrument, the MALDI tandem mass spectrometer offers several unique features, including (1) offline separation to include all of the peptides eluted from LC on a MALDI plate, (2) decoupling of MS and MS/MS acquisition that allows for a selective MS/MS analysis, and (3) an ease to interpret data structure because of the generation of predominantly singly charged ions. Some of the recent applications of MALDI tandem instruments in proteomics and biomedical research include the applications of tandem time-of-flight (TOF/TOF),9,14–28 quadrupole time-of-flight (QqTOF),29–40 and Fourier transform (FT)41–46 mass spectrometers. Quantitative analyses using MALDI tandem MS have also been reported, including isotope-coded affinity tag (ICAT)-based selective quantitative protein profiling in comparison of two proteomes,30,36,47 multiplex quantitative protein profiling of human cerebrospinal fluid (CSF) for neurodegeneration biomarker discovery,5 and targeted analysis of selected N-linked glycosylated peptides/proteins in human serum.9 Here, we report the implementation and application of a LC MALDI TOF/TOF-based platform for targeted quantitative proteomics analysis in human CSF. The issues related to experimental strategy, method robustness, chromatographic separation, and mass spectrometric quantification were also discussed. Finally, a subset of candidate biomarkers, revealed via profiling proteomics and associated with aging and neurodegenerative diseases, were detected and validated using a LC MALDI TOF/TOF-based targeted quantitative proteomics platform.

Experimental Procedures Synthesis of Stable-Isotope-Labeled Peptides. (Fluorenylmethoxy)carbonyl (FMOC)-derivatized stable-isotope monomers containing one 15N and five to six 13C atoms (98%) were from Cambridge Isotope Laboratories (Andover, MA). Preloaded Wang resins were from Applied Biosystems (Foster City, CA).

research articles

Amino acids activated in situ with 1-H-benzotriazolium, 1-[bis(dimethylamino)methylene]-hexafluorophosphate(1-), and 3-oxide/1-hydroxybenzotriazole hydrate were coupled at a 5-fold molar excess over peptide. Each coupling cycle was followed by capping with acetic anhydride to avoid accumulation of oneresidue deletion peptide byproducts. After synthesis, peptide resins were treated with a standard scavenger-containing trifluoroacetic acid–water cleavage solution, and the peptides were precipitated by addition to cold ether. Peptides were purified by reversed-phase C18 high-performance liquid chromatography (HPLC) and characterized by MALDI-TOF (Biflex III, Bruker Daltonics, Billerica, MA) and ion-trap (LCQ DecaXP, ThermoFinnigan, San Jose, CA) mass spectrometers. The purified synthetic peptide stocks were quantified by amino acid analysis using a PicoTag station (Waters, Milford, MA) for acid hydrolysis and an AccQ-Fluor reagent kit (Waters, Milford, MA) for amino acid derivatization. Collection of CSF and Patient Characterization. The study was approved by the Institutional Review Boards of the University of Washington, Baylor College of Medicine, University of California at San Diego, and Oregon Health and Science University, and written informed consent was obtained from patients or their legally authorized representative. All individuals (normal control subjects and patients) underwent an evaluation that consisted of medical history, physical and neurologic examinations, laboratory tests, and neuropsychological assessment. A summary on inclusion and exclusion criteria for normal controls as well as patients with AD and PD has been published elsewhere.5 The collection and characterization of human CSF were performed following wellestablished protocols used in our previous studies, including vigorous control for blood contamination.5,48–50 The age range (average) for middle age normal controls (MA), age-matched normal controls (OC), and patients with early Parkinson’s disease (EPD; H-Y score < 1.5), late Parkinson’s disease (LPD; H-Y score > 3.0), and Alzheimer’s disease (AD) are 43–55 (48), 60–81 (69), 46–70 (60), 58–80 (66), and 61–84 (72), respectively. For each group, the pooled CSF sample was generated by combining an equal volume of individual samples as follows: MA, 22 samples; OC, 29 samples; EPD, 11 samples; LPD, 11 samples; and AD, 10 samples. Sample Preparation and Separation. The protein in 250 µL of pooled CSF was precipitated with 6 volumes of cold acetone overnight, digested with trypsin, and fractionated with strongcation-exchange chromatography (SCX) with a PolySulfethyl A column (2.1 × 200 mm, 5 µm/300 Å, Poly LC, Columbia, MD). The peptides were fractionated and eluted by applying a linear gradient of 0-100% buffer B [5 mM KH2PO4/600 mM KCl/25% acetonitrile (ACN) at pH 3.0] versus buffer A [5 mM KH2PO4/ 25% acetonitrile (ACN) at pH 3.0] with a flow rate at 200 µL/ min. The SCX fractions were combined into six fractions, dried, and resuspended in 0.5% trifluoroacetic acid (TFA) solution, and each sample was spiked with the mixture of reference peptides for a concentration of 2 pmol/250 µL of CSF for each reference peptide. LC/Probot Fractionation and MALDI TOF/TOF Analysis. Each spiked SCX fraction was further separated with reversephase (RP) LC and spotted on the MALDI plate using an Ultimate HPLC system (LC Packing/Dionex, Sunnyvale, CA) coupled with a Probot Micro Fraction collector (LC Packing/ Dionex). A total of 20 µL of each sample was first loaded on a trap column with loading buffer (0.5% TFA/HPLC water) at a flow rate of 0.4 µL/min. The sample was then separated by an Journal of Proteome Research • Vol. 7, No. 2, 2008 721

research articles analytical column (15 cm × 100 µm i.d., Magic C18, 3 µm/100 Å, Michrom Bioresources, Inc., Auburn, CA) using a gradient running from 5 to 50% solvent B (80% ACN/0.1% TFA) versus solvent A (2% ACN/0.1% TFA) for 75 min, with a flow rate at 0.4 µL/min. The effluent from the capillary column was mixed with the recrystallized R-cyano-4-hydroxycinnamic acid matrix solution (7 mg/mL) with a 1:1 ratio in a mixing tee and then deposited onto a stainless MALDI plate with a 576 format (24 × 24). The spotted samples were analyzed by a MALDI TOF/ TOF tandem mass spectrometer (ABI 4700 Proteomics Analyzer, Applied Biosystems, Foster City, CA). Both MS and MS/MS data were acquired with a Nd:YAG laser, with a 200 Hz repetition rate. For MS spectra, 1000 laser shots per spot were used to ensure appropriate ion statistics for quantification. After MS acquisition, an inclusion list containing the selected precursor ions was used for selective MS/MS acquisition. The MS/MS mode was operated with 1 keV collision energy. The CID was performed using air as the collision gas. Typically, 2000 laser shots were used for MS/MS acquisition. Both MS and MS/MS data were acquired using the instrument default calibration. Data Analysis. MS/MS data were searched against the protein database from International Protein Index (IPI) (Human database/version 3.18) from the European Bioinformatics Institute (EBI) and a standard peptide database containing the targeted proteins, using GPS software (Applied Biosystems) with the MASCOT (Matrix Science, Boston, MA) search algorithm. For quantification, triplicate samples were analyzed. The quantification of targeted peptides was accomplished using the abundance ratio of a native peptide to the corresponding stable-isotope-labeled reference peptide, for which the amount of the spike-in was known. For peptides that were eluted across more than one sample spot, the signal intensities from each adjacent spot were summed together to determine a more accurate intensity over the entire peptide elution profile. An in-house developed software was used to automatically calculate the peak intensity ratio of a native peptide to its reference peptide, and the ratios were manually validated. Grouped data were expressed as mean and standard deviation. Changes between groups were analyzed by one-way analysis of variance (ANOVA) using GraphPad Prism 3.0 (San Diego, CA).

Results and Discussion Analytical Flow. The operation of the LC-based MALDI TOF/ TOF mass spectrometer is different from the ESI-based instrument for peptide analysis using a “shotgun” proteomics approach. Some of the differences can be considered as advantages, while others might be described as disadvantages, in part, depending upon the type of experiment performed. One of the unique features of the LC MALDI TOF/TOF approach is that it allows for separable operations on LC separation, MS acquisition, and MS/MS acquisition. Thus, it provides great flexibility in operation modes, either in discovery mode for global protein profiling or browsing mode for targeted peptide/protein analysis,9,30,36,47 and can potentially be operated at high throughput. The implementation of targeted quantitative proteomics on a LC MALDI TOF/TOF platform included two major components: (1) use of synthetic reference peptides with stable isotope labeling as signature markers for identification and quantification of targeted peptides/proteins and (2) high-speed MS analysis and selective MS/MS operation based on the data analysis of MS data. Different from the ESI approach, in which peptides are continually eluted from the LC column and the scan speed is critically important in 722

Journal of Proteome Research • Vol. 7, No. 2, 2008

Pan et al.

Figure 1. Schematic illustration of the analytical flow for a targeted quantitative proteomics analysis using a LC MALDI TOF/ TOF-based platform.

capturing analytes for analysis, the MALDI TOF/TOF instrument allows for the acquisition of MS and MS/MS spectra separately because all of the peptides are separated and deposited on a MALDI plate prior to mass spectrometric analysis. Therefore, information from MS data can be used to generate criteria for selected MS/MS analysis. The analytical flow of the LC MALDI TOF/TOF platform is schematically demonstrated in Figure 1. Briefly, one or multiple peptides from a targeted protein can be selected, chemically synthesized with stable isotope labeling, and spiked in the tryptic-digested sample as signature markers for identification and quantification of the corresponding native peptides. The spiked sample is separated by LC, spotted on a MALDI plate, and interrogated by MALDI TOF/TOF using MS mode. The known masses of the targeted peptides are then searched against the MS data, and only the precursor ions with a mass within the mass search windows are selected for MS/MS acquisition for peptide sequencing. Such MS-based MS/MS analysis provides high selectivity and is well-suited for targeted quantitative proteomics analysis. The quantification of targeted peptides is achieved at the MS level using the intensity ratio of the identified native/reference peptides and the known amount of the corresponding reference peptide spiked in the sample. For complex samples with a great dynamic range in concentration, an optimized, well-balanced separation/spotting strategy, including chromatography gradient, array density, and time interval for spotting, should help to enhance detection

Analysis of Human Cerebrospinal Fluid Using LC MALDI TOF/TOF

research articles

sensitivity and quantification of targeted peptides in a complex system, in particular for those derived from low abundant proteins. Complementary Identification of Targeted Peptides/ Proteins in CSF. As mentioned early, our process of verifying/ validating novel and brain-specific protein markers unique to neurodegeneration has been hampered because of the lack of specific antibodies.5 As an alternative, we tested the feasibility of validating unique peptides identified in human CSF directly with LC MALDI-TOF/TOF-based quantitative proteomics. A total of 14 unique peptides, chemically synthesized with stable isotope labeling, were used as signature references for targeted analysis of the corresponding native peptides in CSF. These peptides were previously observed in our profiling analysis. Because of the complexity of a biological system at the protein level, the selection of appropriate signature peptides to represent a targeted protein is critical for quantitative analysis. Signature peptides can be selected to represent a specific protein or a group of proteins. In general, the criteria to be considered should include the uniqueness of a signature peptide to the corresponding targeted protein or protein group, known post-translational modifications or amino acid variants on the peptide, adequate sensitivity and appropriate mass for the mass spectrometry platform applied (e.g., 800–4000 Da for MALDI TOF/TOF), and good chromatographic characteristics. In this study, we deliberately avoided depletion of abundant proteins, such as albumin and immunoglobulins, prior to proteomics analysis for the concern that other proteins could be depleted along with abundant proteins under nondenaturing conditions. To reduce the analytical difficulties associated with the enormous complexity and dynamic range of protein concentrations of human CSF, the tryptic digest of CSF proteins was fractionated with SCX and combined into six fractions. Each fraction was spiked with a known amount of reference peptides and subjected to LC separation/spotting and MALDI TOF/TOF-targeted analysis. Figure 2 demonstrates the selective detection of targeted peptide ATVVYQGER (β-2-glycoprotein 1 precursor, IPI00298828; the underline indicates the amino acid that was stable-isotopelabeled in the reference peptide) in CSF. After MS acquisition, the mass of the reference peptide was searched against the MS data to locate and identify the precursor ions that have a mass within the mass search window ((0.3 Da). The identification of the reference peptide using accurate mass led to the preliminary identification of the corresponding native peptide, which was defined by a distinct mass shift from the reference peptide because of stable isotope labeling. Notably, for complex samples, the search for the mass of a peptide might result in multiple candidate precursor ions with similar mass but located at different spots on the MALDI plate; therefore, a MS/MS analysis is needed to further distinguish and identify the targeted peptide. The precursor ions selected (including reference and native peptides) were then subjected to MS/MS analysis for peptide sequencing and identification, which provides additional identification confidence on peptide validation in addition to the mass matching. The inset in Figure 2A shows the MS signals of the reference and native peaks. The fourth amino acid of the reference peptide, V, was labeled with 13 C and 15N and introduced a mass difference of 6 Da, which well-distinguished the native peptide versus the reference peptide. The corresponding MS/MS spectra for the identification of the peptide pair are shown in Figure 2B.

Figure 2. Selective detection of peptide ATVVYQGER in CSF using a targeted quantitative proteomics approach. (A) Sample was first acquired in MS mode, and the mass of the targeted reference peptide was searched against the MS data. The mass matching led to the primary identification of the peptide pairs (inset, reference versus native). (B) Selected precursor ions were subjected to MS/MS analysis for peptide sequencing and further identification.

The overall sensitivity of detecting the native peptides in CSF, especially those with low abundance, largely depends upon the concentration of a specific peptide as well as its physicochemical properties for a given sample complexity, experimental conditions, and mass spectrometer used. Two major separation steps were used to reduce the sample complexity in this experiment: SCX and RP LC. The separation mechanism of SCX is primarily based on the electrostatic characteristics of the peptides. For a given column condition, in addition to the overall net charge of a peptide, other physicochemical properties of a peptide, such as hydrophobicity, structural conformation, chain length, charge distribution, and density, could play important roles in determining the retention behavior of the peptide.51–55 In a complex sample, such as the whole proteome of CSF in this study, intermolecular interactions and nonspecific bindings may also cause peptides to be separated with low resolution. Indeed, a few peptides, including ATVVYQGER, were detected in multiple SCX fractions of CSF. Similar issues, with different mechanisms, were also likely involved in RP separation during LC/spotting. For instance, low abundant peptides with higher hydrophobicity are more difficult to be separated and more likely to be lost in the C18 column. Peptides with Pro-Pro bonds can cause peak broadening during RP separation. While many of the chromatography issues can be minimized by optimizing and balancing experimental parameters, others are the inherent characteristics of a specific peptide. Additionally, the physicochemical properties of a peptide play a critical role in determining the mass spectrometric sensitivity and fragmentation pattern for a given ionization method and mass analyzer. For MALDI TOF/TOF, fundamental studies have provided newer models for better interpretation of the mechanism of the primary ionization/desorption process Journal of Proteome Research • Vol. 7, No. 2, 2008 723

research articles 56

in general. However, systematic works focusing on studying the mass spectrometric behavior of peptides in correlating to their physicochemical properties are still limited. Nevertheless, empirical evidence has suggested that the ionization/desorption efficiency varies significantly for peptides with different chemical structure and properties. For instance, we observed that the sensitivity of targeted peptide SSQGGSLPSEEK (32 kDa protein, IPI00647027) with MALDI TOF/TOF was significantly lower than that of targeted peptide EPYPGSAEVIR (neurexin-1 precursor, IPI00442299) with the same concentration. Because the two peptides have similar mass, the intensity of peptide SSQGGSLPSEEK was further suppressed by peptide EPYPGSAEVIR when analyzed together without chromatographic separation. Previous studies have suggested that a peptide signal generated by the MALDI process can be a function of multiple factors, including peptide charge (e.g., arginine),57,58 peptide size and hydrophobicity,57,59–61 and secondary structure.62 The assessment of the detection sensitivity of a peptide in a complex sample can be further complicated by sample complexity, for which ion suppression effects also need to be considered. Taken together, the overall sensitivity for detection of a targeted peptide in a complex proteome is a convolution involving different aspects and critically depends upon the intrinsic property of the peptide and the local ionization/ desorption environment. Recently, empirical-based models have been developed for several different proteomics workflows to evaluate the detection sensitivity of peptides based on their physicochemical properties.63 Such models, although not yet readily available for a LC MALDI TOF/TOF platform, will be useful to facilitate the selection of peptides for targeted quantitative proteomics analysis. Figure 3 shows the spectra of the targeted peptide pairs (reference versus native) that were detected in the whole CSF proteome. The mass differences between the corresponding reference and native peptides were defined by the stable isotope labeling and well-distinguished by the MALDI TOF/ TOF with high resolution. Furthermore, the well-resolved isotope distribution of the signals provided additional identification of the reference peptides, which were chemically synthesized with stable isotope labeling, thus presenting a different isotope distribution pattern. It is notable that, for peptide ALYLQYTDETFR (ceruloplasmin precursor, IPI00017601), additional peaks with similar mass and chromatographic retention time were also observed adjacent to the targeted peptides (Figure 3), suggesting the vast complexity of the sample, even after the two-dimensional separation of SCX and RP LC. Owing to the high resolution and mass accuracy that the TOF mass analyzer (reflector mode) can provide, the interference peaks were resolved from the targeted peptides and the complications for identification and quantification of targeted peptides were minimized. Our experience suggests that the LC MALDI TOF/TOF-based platform is highly selective and specific in detection of targeted peptides within a complex background. Because the identification of a targeted peptide relies on the accurate mass of the peptide and its exact sequence, when appropriate signature peptides are selected, conceptually, the platform is capable of detecting a specific protein isomer or mutation. We have observed that a peptide with an amino acid variant showed a difference in quantification comparing to a different peptide with no amino acid variant, which was also derived from the same protein. If validated, this phenomenon may suggest that peptides with 724

Journal of Proteome Research • Vol. 7, No. 2, 2008

Pan et al. different variants because of gene mutation or polymorphism can be detected and quantified using the same approach. Absolute Quantification of Targeted Peptides/Proteins in CSF. The stable-isotope-labeled peptides spiked in the sample not only served as signature references for peptide/protein identification but also as internal standards for absolute quantification. Because a reference peptide and the corresponding native peptide share the same chromatographic characteristics, conceptually, a reference/native peptide pair should be co-elute from RP LC and deposited on the MALDI plate. Figure 4 shows the reconstructed ion chromatogram of peptide EPYPGSAEVIR (neurexin-1 precursor, IPI00442299), in which both the reference and native peptides eluted across spot 210-216. The ratio of integrated peak intensity (cluster area) of the peptide pair was used to quantify the concentration of native peptide in the CSF. In quantifying the peptides, it is important to note that during the chromatography separation a peptide can be eluted across more than one spot on a MALDI plate depending upon the concentration of the peptide, physicochemical properties, and the experimental settings. In Figure 5, the peptide pair of ATVVYQGER (β-2-glycoprotein 1 precursor, IPI00298828) was detected in consecutive multiple spots with various peak intensities. Such phenomenon needs to be considered in quantifying the targeted peptides, especially when there is a significant difference in abundance between the reference and native peptide. Thus, the peak intensities of a peptide eluting across multiple spots were integrated to improve the accuracy of quantification. Calibration curve for each targeted peptide was generated using synthetic reference peptides with and without stable isotope labeling. The synthetic peptides with no stable isotope labeling were used to represent the “native” targeted peptides in construction of the calibration curve. The standards were prepared as follows. Each standard contained the stable isotopic reference peptides with a fixed concentration at 2 pmol/µL each and the corresponding “native” peptides in a series of dilutions of 0.5, 1, 2, and 5 pmol/µL. In each calibration curve, the intensity ratio of a native (no labeling)/reference (stable isotope labeling) peak pair directly reflects the amount of the native peptide in a unit volume spiked with 2 pmol of corresponding isotope-labeled peptide. With 250 µL of CSF to start with and a total of 2 pmol of each reference peptide to spike in, the reading on the calibration curve, therefore, directly quantifies the amount of native peptides detected in 250 µL (unit volume) of CSF. The calibration curves of the five peptides measured in the pooled CSF of MA were demonstrated in Figure 6. On the basis of the calibration curve, the absolute concentrations of the peptides detected in the pooled MA CSF were calculated (Table 1) using the ratio of the integrated peak intensity of a native peptide versus the corresponding reference peptide, for which its concentration was known. MALDI TOF/ TOF provides superior mass accuracy and resolution for peak identification and isotope resolution, which facilitated the identification confidence and quantification accuracy of targeted peptides in complex human CSF. As an emerging technique, the future improvement of software automation for peptide quantification is expected to significantly enhance the throughput and robustness of the MALDI TOF/TOF platform for targeted proteomics analysis. Detection of Targeted Peptides in CSF of AD, PD, and Controls. The same mass spectrometric platform and analytical flow were applied to detect a group of targeted peptides in the

Analysis of Human Cerebrospinal Fluid Using LC MALDI TOF/TOF

research articles

Figure 3. Identification of targeted peptides and the corresponding proteins in CSF. The synthetic reference peptides served as signature markers as well as internal standards for identification and quantification of the corresponding native peptides. Note that the amino acid underlined indicates the amino acid that was stable-isotope-labeled in reference peptides.

pooled CSF from patients with early Parkinson’s disease (EPD), late Parkinson’s disease (LPD), and Alzheimer’s disease (AD) in comparing to age-matched control (OC) as well as middle age control (MA). Among the 14 peptides tested, the peptides corresponding to three proteins, i.e., neurexin-1, β-2-glycoprotein 1, and R-1-acid glycoprotein, were consistently detected with quantitative changes among the samples. All three proteins, neurexin-1, R-1-acid glycoprotein, and β-2-glycoprotein

1, are glycoproteins and have been identified in our previous CSF glycoprotein study.50 Neurexin-1 is a neuronal cell-surface adhesion protein that may be involved in cell recognition and mediating intercellular signaling. There are three isoforms of this protein: isoform 1 and 2 of neurexin-1-R and neurexin-1-β, all containing the peptide EPYPGSAEVIR. Thus, quantitative analysis of the neurexin-1 precursor using target peptide (EPYPGSAEVIR) Journal of Proteome Research • Vol. 7, No. 2, 2008 725

research articles

Figure 4. Elution profile of peptide pair EPYPGSAEVIR (neurexin-1 precursor). Both the stable-isotope-labeled reference peptide and the native peptide co-eluted across spot position 210–216. The ratio of integrated peak intensities (cluster area) was used to quantify the native peptide in CSF.

Pan et al. system and inflammation during the acute phase reaction.66 This protein was observed upregulated in AD patients but decreased in PD patients (Figure 7B), consistent with our previous profiling investigation.5 In particular, the significant overexpression (p < 0.05) of this protein in AD may suggest neuroinflammation, which was reported as a possible essential cofactor implicating with AD,67 might be involved with some or all of the AD patients included in this study. β-2-glycoprotein 1 is also known as apolipoprotein H because of its phospholipid-binding function. In our previous study, this protein increased and decreased in AD and PD CSF, respectively, compared to age-matched controls.5 Apparently, a decrease in β-2-glycoprotein in PD patients was not validated in the current study. One of the possibilities underlying the discrepancy could be the difference in patients/subjects studied. Nonetheless, a significant upregulation (p < 0.05) of β-2glycoprotein 1 in AD patients was observed using targeted quantitative proteomics (Figure 7C), validating that this protein is indeed increased in most of AD patients. Currently, there is no direct evidence in the literature linking β-2-glycoprotein 1 (apolipoprotein H) to AD. We have speculated that a dramatic increase in β-2-glycoprotein 1 in AD CSF could be a result of physiological/pathological alternation in the brain blood barrier (BBB) or changes in clearance of this protein in AD patients. In addition to further validating this protein in a larger cohort of patients/subjects in the future as a diagnostic marker, we believe that it is important to study the functions of this protein in the CNS, because β-2-glycoprotein has been suggested to be critical in lipid metabolism and regulation of the coagulation system related to thrombotic diseases.68 Indeed, microvascular disease, with microinfarcts, has been reported as a major component of a subset of dementing patients, including those with AD.69

Conclusions Figure 5. Multispot elution of a peptide pair. The peptide pair (reference and native) of ATVVYQGER (β-2-glycoprotein 1 precursor) was eluted across multiple spots, from spot position 128 to 132.

indicated the total abundance of this protein in pooled CSF. Figure 7A shows the total abundance of neurexin-1 precursor, which has apparently decreased in the aged subjects, including OC, EPD, LPD, and AD, compared to MA. Previous studies have suggested that neurexin-1 R might play an important role as one of the two presynaptic receptors binding to R-latrotoxin to trigger massive neurotransmitter release from nerve terminals.64,65 Thus, our observation may imply a possible attenuation on neuron synapse activity as age grows. In comparing the disease groups (EPD, LPD, and AD) to OC, the decrease of neurexin-1 in AD was consistent with our previous study.5 However, the previous observation of the increase of neurexin-1-R precursor in PD5 was not validated in this study. At least two possibilities could explain this discrepancy: (1) different cohort of subjects were used, and (2) target peptide EPYPGSAEVIR measured the total concentration of all three neurexin-1 isoforms rather than just that of neurexin-1-R. The possible implication of neurexin-1-R precursor in PD warrants further investigation, particularly because it decreased in LPD as compared to EPD, meaning that it may serve as a PD progression marker. R-1-Acid glycoprotein is one of the major serum glycoproteins that are involved in modulating the activity of the immune 726

Journal of Proteome Research • Vol. 7, No. 2, 2008

In this study, we demonstrated the application of a LC MALDI TOF/TOF-based platform for selected identification and quantification of a panel of targeted peptides/proteins in human CSF. One of the key features of this method is the complementary approach to searching and identifying native peptides with their corresponding isotopic-labeled counterparts as references. Multiple criteria were applied for searching and identifying targeted peptides, including accurate mass, isotope pattern, and MS/MS analysis for peptide sequencing. The physical locations of targeted peptides on the MALDI plate can also be identified and employed for further analysis of targeted peptides. Such a complementary approach provided high confidence in peptide identification and is extremely important for targeted analysis on a complex sample, such as CSF or blood. Quantification is another important aspect for targeted proteomics analysis. Many issues, as we discussed, might impact the accurate determination of the quantification of targeted peptides. A well-designed sample preparation strategy can enhance the separation of peptides and minimize the number of spots that a peptide elutes across. Furthermore, the accuracy and reproducibility of a mass spectrometric measurement relies on the sensitivity of a peptide for a certain mass spectrometric platform. The selection of unique peptides to represent a targeted protein appeared to play a critical role in quantification, meaning that technical as well as biological factors need to be included in the selection of signature peptides for targeted proteins. Our observation suggested that

research articles

Analysis of Human Cerebrospinal Fluid Using LC MALDI TOF/TOF

Figure 6. Calibration curves of the targeted peptides. The y axis indicates the peak intensity (cluster area) ratio of a native peptide (isotopic light) to the corresponding reference peptide (isotopic heavy). The x axis indicates the concentration of a native peptide in a unit volume, which was spiked with 2 pmol of the corresponding reference peptide. In this case, the unit volume is 250 µL of CSF. Table 1. Quantification of Targeted Peptides/Proteins in Pooled Human CSF from Middle Age Subjectsa intensity ratio (native/reference) protein name

β-2-glycoprotein 1 precursor ceruloplasmin precursor 32 kDa protein neurexin-1 precursor R-1-acid glycoprotein 1 precursor a

monoisotopicion mass (H) (Da)

monoisotopicion mass (L) (Da)

IPI00298828 ATVVYQGER

1028.28

1022.52

1.56

0.18

9.0

12

IPI00017601 ALYLQYTDETFR

1526.49

1519.74

3.62

0.55

16.3

15

IPI00647027 SSQGGSLPSEEK IPI00442299 EPYPGSAEVIR

1212.31 1223.37

1205.56 1217.61

7.27 0.39

1.63 0.02

12.4 6.1

22 5

IPI00022429 EQLGEFYEALDCLR

1749.56

1742.81

26.33

5.50

90.4

21

IPI number

peptide sequence

mean

standard deviation

concentration (pmol/µL)

CV %

The amino acid underlined indicates the amino acid that was stable-isotope-labeled in the reference peptide.

peptides with amino acid variants as a result of genomic mutation or polymorphism might provide different quantitative results. We have discussed already many analytical issues associated with sample complexity and experimental designs both specific

to our application and more generally for targeted quantitative proteomics. In validating the targeted peptides in PD and AD samples compared to middle age and age-matched controls, the neurexin-1 precursor was observed downregulated in aged samples, implying a possible age-related attenuation on neuron Journal of Proteome Research • Vol. 7, No. 2, 2008 727

research articles

Figure 7. Targeted quantitative detection of selected peptides/ proteins in CSFs from middle age (MA) subjects, age-matched control (OC) subjects, earlier PD (EPD) patients, late PD (LPD) patients, and AD patients. (A) Neurexin-1, (B) R-1-acid glycoprotein, and (C) β-2-glycoprotein 1.

synapse activity. R-1-Acid glycoprotein was observed significantly upregulated in AD, suggesting its possible implication with the disease. The observation of an elevated expression of β-2-glycoprotein 1 in AD provides new questions and hypotheses for the role of this protein in implicating AD. To further establish these proteins as potential biomarkers for aging or neurodegenerative diseases, a large cohort of independent samples will be required to assess their specificity and sensitivity, which is part of our ongoing study. It is our expectation that this study will provide useful knowledge for the application of targeted quantitative proteomics for biomarker studies in human CSF and eventually benefit the development of massspectrometry-based high-throughput systems for clinical applications.

Acknowledgment. This research was supported by NIH grants (AG025327, AG05136, NS060252, and ES012703), NIA AG05136, as well as grants from the Michael J. Fox Foundation, Shaw Endowment, Friends of Alzheimer’s Research, Alzheimer’s Association of Western and Central Washington, an anonymous foundation, and the Department of Veterans Affairs. We also deeply appreciate those who have donated their CSF for our studies. References (1) Aebersold, R. Constellations in a cellular universe. Nature 2003, 422 (6928), 115–116.

728

Journal of Proteome Research • Vol. 7, No. 2, 2008

Pan et al. (2) Rifai, N.; Gillette, M. A.; Carr, S. A. Protein biomarker discovery and validation: The long and uncertain path to clinical utility. Nat. Biotechnol. 2006, 24 (8), 971–983. (3) Pang, S.; Smith, J.; Onley, D.; Reeve, J.; Walker, M.; Foy, C. A comparability study of the emerging protein array platforms with established ELISA procedures. J. Immunol. Methods 2005, 302 (1– 2), 1–12. (4) Schwenk, J. M.; Lindberg, J.; Sundberg, M.; Uhlen, M.; Nilsson, P. Determination of binding specificities in highly multiplexed beadbased assays for antibody proteomics. Mol. Cell. Proteomics 2007, 6 (1), 125–132. (5) Abdi, F.; Quinn, J. F.; Jankovic, J.; McIntosh, M.; Leverenz, J. B.; Peskind, E.; Nixon, R.; Nutt, J.; Chung, K.; Zabetian, C.; Samii, A.; Lin, M.; Hattan, S.; Pan, C.; Wang, Y.; Jin, J.; Zhu, D.; Li, G. J.; Liu, Y.; Waichunas, D.; Montine, T. J.; Zhang, J. Detection of biomarkers with a multiplex quantitative proteomic platform in cerebrospinal fluid of patients with neurodegenerative disorders. J. Alzheimer’s Dis. 2006, 9 (3), 293–348. (6) Aebersold, R.; Anderson, L.; Caprioli, R.; Druker, B.; Hartwell, L.; Smith, R. Perspective: A program to improve protein biomarker discovery for cancer. J. Proteome Res. 2005, 4 (4), 1104–1109. (7) Anderson, L. Candidate-based proteomics in the search for biomarkers of cardiovascular disease. J. Physiol. 2005, 563 (part 1), 23–60. (8) Gerber, S. A.; Rush, J.; Stemman, O.; Kirschner, M. W.; Gygi, S. P. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (12), 6940–6945. (9) Pan, S.; Zhang, H.; Rush, J.; Eng, J.; Zhang, N.; Patterson, D.; Comb, M. J.; Aebersold, R. High throughput proteome screening for biomarker detection. Mol. Cell. Proteomics 2005, 4 (2), 182–190. (10) Anderson, N. L.; Anderson, N. G.; Haines, L. R.; Hardie, D. B.; Olafson, R. W.; Pearson, T. W. Mass spectrometric quantitation of peptides and proteins using stable isotope standards and capture by anti-peptide antibodies (SISCAPA). J. Proteome Res. 2004, 3, 235–244. (11) Anderson, L.; Hunter, C. L. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins. Mol. Cell. Proteomics 2006, 5 (4), 573–588. (12) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Protein and polymer analyses up to m/z 100,000 by laser ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 1988, 2, 151–153. (13) Karas, M.; Hillenkamp, F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal. Chem. 1988, 60, 2299–2301. (14) Shui, W.; Liu, Y.; Fan, H.; Bao, H.; Liang, S.; Yang, P.; Chen, X. Enhancing TOF/TOF-based de novo sequencing capability for high throughput protein identification with amino acid-coded mass tagging. J. Proteome Res. 2005, 4 (1), 83–90. (15) Samyn, B.; Debyser, G.; Sergeant, K.; Devreese, B.; Van Beeumen, J. A case study of de novo sequence analysis of N-sulfonated peptides by MALDI TOF/TOF mass spectrometry. J. Am. Soc. Mass Spectrom. 2004, 15 (12), 1838–1852. (16) Zhen, Y.; Xu, N.; Richardson, B.; Becklin, R.; Savage, J. R.; Blake, K.; Peltier, J. M. Development of a LC-MALDI method for the analysis of protein complexes. J. Am. Soc. Mass Spectrom. 2004, 15 (6), 803–822. (17) Rejtar, T.; Chen, H. S.; Andreev, V.; Moskovets, E.; Karger, B. L. Increased identification of peptides by enhanced data processing of high-resolution MALDI TOF/TOF mass spectra prior to database searching. Anal. Chem. 2004, 76 (20), 6017–6028. (18) Wuhrer, M.; Hokke, C. H.; Deelder, A. M. Glycopeptide analysis by matrix-assisted laser desorption/ionization tandem time-offlight mass spectrometry reveals novel features of horseradish peroxidase glycosylation. Rapid Commun. Mass Spectrom. 2004, 18 (15), 1741–1748. (19) Zhu, X.; Papayannopoulos, I. A. Improvement in the detection of low concentration protein digests on a MALDI TOF/TOF workstation by reducing R-cyano-4-hydroxycinnamic acid adduct ions. J. Biomol. Technol. 2003, 14 (4), 298–307. (20) Morelle, W.; Slomianny, M. C.; Diemer, H.; Schaeffer, C.; van Dorsselaer, A.; Michalski, J. C. Fragmentation characteristics of permethylated oligosaccharides using a matrix-assisted laser desorption/ionization two-stage time-of-flight (TOF/TOF) tandem mass spectrometer. Rapid Commun. Mass Spectrom. 2004, 18 (22), 2637–2649. (21) Stephens, E.; Maslen, S. L.; Green, L. G.; Williams, D. H. FragmentationcharacteristicsofneutralN-linkedglycansusingaMALDI-TOF/ TOF tandem mass spectrometer. Anal. Chem. 2004, 76 (8), 2343– 2354.

Analysis of Human Cerebrospinal Fluid Using LC MALDI TOF/TOF (22) Bienvenut, W. V.; Deon, C.; Pasquarello, C.; Campbell, J. M.; Sanchez, J. C.; Vestal, M. L.; Hochstrasser, D. F. Matrix-assisted laser desorption/ionization-tandem mass spectrometry with high resolution and sensitivity for identification and characterization of proteins. Proteomics 2002, 2 (7), 868–876. (23) Yergey, A. L.; Coorssen, J. R.; Backlund, P. S., Jr.; Blank, P. S.; Humphrey, G. A.; Zimmerberg, J.; Campbell, J. M.; Vestal, M. L. De novo sequencing of peptides using MALDI/TOF-TOF. J. Am. Soc. Mass Spectrom. 2002, 13 (7), 784–791. (24) Chen, H. S.; Rejtar, T.; Andreev, V.; Moskovets, E.; Karger, B. L. Enhanced characterization of complex proteomic samples using LC-MALDI MS/MS: Exclusion of redundant peptides from MS/ MS analysis in replicate runs. Anal. Chem. 2005, 77 (23), 7816– 7825. (25) Gu, X.; Deng, C.; Yan, G.; Zhang, X. Capillary array reversed-phase liquid chromatography-based multidimensional separation system coupled with MALDI-TOF-TOF-MS detection for high-throughput proteome analysis. J. Proteome Res. 2006, 5 (11), 3186–3196. (26) Qualtieri, A.; Urso, E.; Le Pera, M.; Scornaienchi, M.; Quattrone, A.; Di Donna, L.; Napoli, A.; Sindona, G. Proteomics of bovine myelin sheath: Characterization of a truncated form of P0 by MALDI-TOF/TOF mass spectrometry. J. Am. Soc. Mass Spectrom. 2006, 17 (2), 117–123. (27) Samyn, B.; Sergeant, K.; Memmi, S.; Debyser, G.; Devreese, B.; Van Beeumen, J. MALDI-TOF/TOF de novo sequence analysis of 2D PAGE-separated proteins from Halorhodospira halophila, a bacterium with unsequenced genome. Electrophoresis 2006, 27 (13), 2702–2711. (28) Ji, C.; Li, L. Quantitative proteome analysis using differential stable isotopic labeling and microbore LC-MALDI MS and MS/MS. J. Proteome Res. 2005, 4 (3), 734–742. (29) Nakanishi, T.; Ohtsu, I.; Furuta, M.; Ando, E.; Nishimura, O. Direct MS/MS analysis of proteins blotted on membranes by a matrixassisted laser desorption/ionization-quadrupole ion trap-timeof-flight tandem mass spectrometer. J. Proteome Res. 2005, 4 (3), 743–747. (30) Griffin, T. J.; Lock, C. M.; Li, X. J.; Patel, A.; Chervetsova, I.; Lee, H.; Wright, M. E.; Ranish, J. A.; Chen, S. S.; Aebersold, R. Abundance ratio-dependent proteomic analysis by mass spectrometry. Anal. Chem. 2003, 75 (4), 867–874. (31) Koy, C.; Mikkat, S.; Raptakis, E.; Sutton, C.; Resch, M.; Tanaka, K.; Glocker, M. O. Matrix-assisted laser desorption/ionizationquadrupole ion trap-time of flight mass spectrometry sequencing resolves structures of unidentified peptides obtained by in-gel tryptic digestion of haptoglobin derivatives from human plasma proteomes. Proteomics 2003, 3 (6), 851–858. (32) Raska, C. S.; Parker, C. E.; Sunnarborg, S. W.; Pope, R. M.; Lee, D. C.; Glish, G. L.; Borchers, C. H. Rapid and sensitive identification of epitope-containing peptides by direct matrix-assisted laser desorption/ionization tandem mass spectrometry of peptides affinity-bound to antibody beads. J. Am. Soc. Mass Spectrom. 2003, 14 (10), 1076–1085. (33) Raska, C. S.; Parker, C. E.; Dominski, Z.; Marzluff, W. F.; Glish, G. L.; Pope, R. M.; Borchers, C. H. Direct MALDI-MS/MS of phosphopeptides affinity-bound to immobilized metal ion affinity chromatography beads. Anal. Chem. 2002, 74 (14), 3429–3433. (34) Verhaert, P.; Uttenweiler-Joseph, S.; de Vries, M.; Loboda, A.; Ens, W.; Standing, K. G. Matrix-assisted laser desorption/ionization quadrupole time-of-flight mass spectrometry: An elegant tool for peptidomics. Proteomics 2001, 1 (1), 118–131. (35) Chernushevich, I. V.; Loboda, A. V.; Thomson, B. A. An introduction to quadrupole-time-of-flight mass spectrometry. J. Mass Spectrom. 2001, 36 (8), 849–865. (36) Griffin, T. J.; Gygi, S. P.; Rist, B.; Aebersold, R.; Loboda, A.; Jilkine, A.; Ens, W.; Standing, K. G. Quantitative proteomic analysis using a MALDI quadrupole time-of-flight mass spectrometer. Anal. Chem. 2001, 73 (5), 978–986. (37) Kirpekar, F.; Krogh, T. N. RNA fragmentation studied in a matrixassisted laser desorption/ionisation tandem quadrupole/orthogonal time-of-flight mass spectrometer. Rapid Commun. Mass Spectrom. 2001, 15 (1), 8–14. (38) Loboda, A. V.; Krutchinsky, A. N.; Bromirski, M.; Ens, W.; Standing, K. G. A tandem quadrupole/time-of-flight mass spectrometer with a matrix-assisted laser desorption/ionization source: Design and performance. Rapid Commun. Mass Spectrom. 2000, 14 (12), 1047– 1057. (39) Shevchenko, A.; Loboda, A.; Ens, W.; Standing, K. G. MALDI quadrupole time-of-flight mass spectrometry: A powerful tool for proteomic research. Anal. Chem. 2000, 72 (9), 2132–2141. (40) Krokhin, O. V.; Ens, W.; Standing, K. G. MALDI QqTOF MS combined with off-line HPLC for characterization of protein

(41)

(42)

(43)

(44)

(45)

(46)

(47)

(48)

(49) (50)

(51)

(52) (53) (54)

(55)

(56) (57) (58) (59) (60)

research articles

primary structure and post-translational modifications. J. Biomol. Technol. 2005, 16 (4), 429–440. Brock, A.; Horn, D. M.; Peters, E. C.; Shaw, C. M.; Ericson, C.; Phung, Q. T.; Salomon, A. R. An automated matrix-assisted laser desorption/ionization quadrupole Fourier transform ion cyclotron resonance mass spectrometer for “bottom-up” proteomics. Anal. Chem. 2003, 75 (14), 3419–3428. Moyer, S. C.; Budnik, B. A.; Pittman, J. L.; Costello, C. E.; O’Connor, P. B. Attomole peptide analysis by high-pressure matrix-assisted laser desorption/ionization Fourier transform mass spectrometry. Anal. Chem. 2003, 75 (23), 6449–6454. Albach, C.; Damoc, E.; Denzinger, T.; Schachner, M.; Przybylski, M.; Schmitz, B. Identification of N-glycosylation sites of the murine neural cell adhesion molecule NCAM by MALDI-TOF and MALDI-FTICR mass spectrometry. Anal. Bioanal. Chem. 2004, 378 (4), 1129–1135. Andre, M.; Le Caer, J. P.; Greco, C.; Planchon, S.; El Nemer, W.; Boucheix, C.; Rubinstein, E.; Chamot-Rooke, J.; Le Naour, F. Proteomic analysis of the tetraspanin web using LC-ESI-MS/MS and MALDI-FTICR-MS. Proteomics 2006, 6 (5), 1437–1449. Becker, J. S.; Zoriy, M.; Pickhardt, C.; Damoc, E.; Juhacz, G.; Palkovits, M.; Przybylski, M. Determination of phosphorus-, copper-, and zinc-containing human brain proteins by LA-ICPMS and MALDI-FTICR-MS. Anal. Chem. 2005, 77 (18), 5851–5860. Tanaka, K.; Takenaka, S.; Tsuyama, S.; Wada, Y. Determination of unique amino acid substitutions in protein variants by peptide mass mapping with FT-ICR MS. J. Am. Soc. Mass Spectrom. 2006, 17 (4), 508–513. Griffin, T. J.; Han, D. K.; Gygi, S. P.; Rist, B.; Lee, H.; Aebersold, R.; Parker, K. C. Toward a high-throughput approach to quantitative proteomic analysis: Expression-dependent protein identification by mass spectrometry. J. Am. Soc. Mass Spectrom. 2001, 12 (12), 1238–1246. Zhang, J.; Goodlett, D. R.; Quinn, J. F.; Peskind, E.; Kaye, J. A.; Zhou, Y.; Pan, C.; Yi, E.; Eng, J.; Wang, Q.; Aebersold, R. H.; Montine, T. J. Quantitative proteomics of cerebrospinal fluid from patients with Alzheimer disease. J. Alzheimer’s Dis. 2005, 7 (2), 125–133, discussion 173–180. Xu, J.; Chen, J.; Peskind, E. R.; Jin, J.; Eng, J.; Pan, C.; Montine, T. J.; Goodlett, D. R.; Zhang, J. Characterization of proteome of human cerebrospinal fluid. Int. Rev. Neurobiol. 2006, 73, 29–98. Pan, S.; Wang, Y.; Quinn, J. F.; Peskind, E. R.; Waichunas, D.; Wimberger, J. T.; Jin, J.; Li, J. G.; Zhu, D.; Pan, C.; Zhang, J. Identification of glycoproteins in human cerebrospinal fluid with a complementary proteomic approach. J. Proteome Res. 2006, 5 (10), 2769–2779. Shen, Y.; Jacobs, J. M.; Camp, D. G.; Fang, R.; Moore, R. J.; Smith, R. D.; Xiao, W.; Davis, R. W.; Tompkins, R. G. Ultra-high-efficiency strong cation exchange LC/RPLC/MS/MS for high dynamic range characterization of the human plasma proteome. Anal. Chem. 2004, 76 (4), 1134–1144. Crimmins, D. L.; Gorka, J.; Thoma, R. S.; Schwartz, B. D. Peptide characterization with a sulfoethyl aspartamide column. J. Chromatogr. 1988, 443, 63–71. Alpert, A. J.; Andrews, P. C. Cation-exchange chromatography of peptides on poly(2-sulfoethyl aspartamide)-silica. J. Chromatogr. 1988, 443, 85–96. Burke, T. W.; Mant, C. T.; Black, J. A.; Hodges, R. S. Strong cationexchange high-performance liquid chromatography of peptides. Effect of non-specific hydrophobic interactions and linearization of peptide retention behaviour. J. Chromatogr. 1989, 476, 377– 389. Kennedy, L. A.; Kopaciewicz, W.; Regnier, F. E. Multimodal liquid chromatography columns for the separation of proteins in either the anion-exchange or hydrophobic-interaction mode. J. Chromatogr. 1986, 359, 73–84. Karas, M.; Kruger, R. Ion formation in MALDI: The cluster ionization mechanism. Chem. Rev. 2003, 103 (2), 427–440. Krause, E.; Wenschuh, H.; Jungblut, P. R. The dominance of arginine-containing peptides in MALDI-derived tryptic mass fingerprints of proteins. Anal. Chem. 1999, 71 (19), 4160–4165. Valero, M.-L.; Giralt, E.; Andreu, D. An investigation of residuespecific contributions to peptide desorption in MALDI-TOF mass spectrometry. Lett. Pept. Sci. 1999, 6, 109–115. Cohen, S. L.; Chait, B. T. Influence of matrix solution conditions on the MALDI-MS analysis of peptides and proteins. Anal. Chem. 1996, 68 (1), 31–37. Olumee, Z.; Sadeghi, M.; Tang, X.; Vertes, A. Amino acid composition and wavelength effects in matrix-assisted laser desorption/ ionization. Rapid Commun. Mass Spectrom. 1995, 9, 744–752.

Journal of Proteome Research • Vol. 7, No. 2, 2008 729

research articles (61) Amado, F. M. L.; Domingues, P.; Santana-Marques, M. G.; FerrerCorreia, A. J.; Tomer, K. B. Discrimination effects and sensitivity variations in matrix-assisted laser desorption/ionization. Rapid Commun. Mass Spectrom. 1997, 11, 1347–1352. (62) Wenschuh, H.; Halada, P.; Lamer, S.; Jungblut, P.; Krause, E. The ease of peptide detection by matrix-assisted laser desorption/ionization mass spectrometry: The effect of secondary structure on signal intensity. Rapid Commun. Mass Spectrom. 1998, 12 (3), 115–119. (63) Mallick, P.; Schirle, M.; Chen, S. S.; Flory, M. R.; Lee, H.; Martin, D.; Ranish, J.; Raught, B.; Schmitt, R.; Werner, T.; Kuster, B.; Aebersold, R. Computational prediction of proteotypic peptides for quantitative proteomics. Nat. Biotechnol. 2007, 25 (1), 125–131. (64) Tobaben, S.; Sudhof, T. C.; Stahl, B. Genetic analysis of R-latrotoxin receptors reveals functional interdependence of CIRL/latrophilin 1 and neurexin 1 R. J. Biol. Chem. 2002, 277 (8), 6359–6365. (65) Hlubek, M.; Tian, D.; Stuenkel, E. L. Mechanism of R-latrotoxin action at nerve endings of neurohypophysis. Brain Res. 2003, 992 (1), 30–42.

730

Journal of Proteome Research • Vol. 7, No. 2, 2008

Pan et al. (66) Hochepied, T.; Berger, F. G.; Baumann, H.; Libert, C. R1-acid glycoprotein: An acute phase protein with inflammatory and immunomodulating properties. Cytokine Growth Factor Rev. 2003, 14 (1), 25–34. (67) Finch, C. E.; Morgan, T. E. Systemic inflammation, infection, ApoE alleles, and Alzheimer disease: A position paper. Curr. Alzheimer Res. 2007, 4 (2), 185–189. (68) Najmey, S. S.; Keil, L. B.; Adib, D. Y.; DeBari, V. A. The association of antibodies to β 2 glycoprotein I with the antiphospholipid syndrome: A meta-analysis. Ann. Clin. Lab. Sci. 1997, 27 (1), 41– 46. (69) White, L.; Petrovitch, H.; Hardman, J.; Nelson, J.; Davis, D. G.; Ross, G. W.; Masaki, K.; Launer, L.; Markesbery, W. R. Cerebrovascular pathology and dementia in autopsied Honolulu-Asia aging study participants. Ann. N.Y. Acad. Sci. 2002, 977, 9–23.

PR700630X