Shotgun Sequencing of the Human CerebrospinalFluid Proteome Using 2D LC/MS with theFinnigan ProteomeX LTQAndreas F. Hühmer1, R.G. Biringer1, H. Amato1, M.G. Harrington2

1Thermo Electron Corporation, San Jose, CA, USA; 2Huntington Medical Research Institutes, Pasadena, CA, USA

ApplicationNote: 342

Key Words

• Finnigan™

ProteomeX LTQ™

• Cerebrospinalfluid

• MudPIT

• MultidimensionalChromatography

• Sub fmolesensitivity

Introduction

Human cerebrospinal fluid (CSF) is in direct contact withthe extra-cellular space of the brain and it contains acomplex collection of small molecules, peptides, proteins,and protein fragments that reflect biochemical processesin the brain. Indepth analysis of the CSF proteome shouldprovide a readily accessible window into the health stateof the central nervous system (CNS). It was demonstratedrecently that diseases involving the CNS markedly andcharacteristically alter the concentrations and isoformpatterns of CSF proteins (Andreasen et al, 1999).

Migraine is the most common neurological conditionin the developed world, effecting 10% of the population.The pathophysiological processes of migraines are notwell understood, and existing hypotheses cannot connectall of the known facts within a unified framework. Thismeans that, though there is a wide range of effectivetreatments available, treatment that is successful for onepatient may not have an effect on another. One possibleapproach to study migraine would be to evaluate the CSFto look for biochemical changes taking place in the brainduring and after a migraine attack.

We have recently used 2D-PAGE methods in combina-tion with nanospray LC-MS/MS to identify more than 40proteins in CSF in order to determine significant changesin protein levels associated with acute migraine attacks(Harrington et al, 2003). It is expected that many proteinsthat play a role in migraine will be present at low concen-tration and a more sensitive analysis method is required tofind other relevant changes. Analysis of CSF using on-line,multidimensional LC-MS/MS (2D LC/MS) provides anopportunity for the detection and quantification of lowerabundance proteins.

Goal

This study utilizes a Finnigan ProteomeX LTQ to performautomated 2D LC/MS to identify low-abundance proteinsfrom CSF.

Experimental Conditions

Sample Prep: A sample of CSF (135 µg total protein)was dissolved in 6 M urea and reduced with dithiothreitol(DTT, 30 µg) at room temperature for one hour. 100 µgof iodoacetamide was added and incubated at room tem-perature in the dark for one hour. Excess iodoacetamidewas removed by adding 80 µg of DTT, followed by incu-bation at room temperature for one hour. Excess reagentswere removed and the buffer exchanged (3x) to 100 mMammonium bicarbonate (pH 8) with spin filtration(Vivaspin® 500) to a final volume of 245 µL. 5.4 µg ofa proteolytic enzyme (Princeton Separations, Inc.) wasadded and the solution incubated overnight at 37°C. Thereaction was quenched by adding 5 µL of formic acid thefollowing morning.

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LC Separation and MS Analysis

Automated, on-line 2D LC/MS was performed using aFinnigan ProteomeX LTQ workstation.

HPLC Conditions

HPLC System: Two Finnigan Surveyor™ MS pumpswith flow splitters

Column 1: Strong cation exchange (100 ×0.32 mm 5 µm, BioBasic™ SCX)

Capture Column(s): Two C18 reversed phase nanotrapcolumns (Zorbax® 5 × 0.3 mm,Agilent Technologies, Palo Alto, CA)

Column 2: PicoFrit™ nanobore HPLC columnwith 15 µM ID pulled tip (BioBasic5 µm 100 mm × 75 µm, NewObjective, Inc, Cambridge, MA)

Flow Rate: 220 nL/min

Mobile Phase: A: Water with 0.1% Formic Acid

B: 100% Acetonitrile with 0.1%Formic Acid

Ion ExchangeGradient: Stepwise elution from SCX column

to C18 capture columns(s), with20 mL of NH4Cl at the followingconcentrations: 0, 10, 20, 40, 60, 80,100, 120, 150, 200, 400, 800 mM

Reversed PhaseGradient: 0–60% B over 180 minutes

MS ConditionsMS System: Finnigan LTQ linear ion trap mass

spectrometer (Figures 1 and 2)Ionization Mode: Positive ion mode, nanosprayScan Sequence: Full-scan MS, five Data Dependent™

MS/MS scansMass Range: 450–1600 DaDynamic Exclusion™

Settings: Repeat count, 1 Repeat duration,0.5 min Exclusion duration, 3.0 min

z

x Detector 1

Detector 2

hybridization

Figure 1: Schematic of the Finnigan LTQ linear ion trap mass spectrometer. Dual detectors present on both sides of the linear trap allow detec-tion of nearly 100% of the ejected ions. Ions can also be ejected axially to a second detector, such as an FT-ICR cell on the Finnigan LTQ FT™.

Figure 2: Linear ion trap structure, showing side slits for exit of ionsand 100% detection by two detectors.

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System Configuration

Figure 3 shows the plumbing configuration for theanalysis. After the digested protein was injected onto theion exchange column, increasing concentrations of NH4Clwere injected (20 mL/injection) through the SCX columnto elute different populations of peptides, varying by netsurface charge. Each step of elution transferred thematerial to a short, reversed phase capture column fordesalting. Peptides were subsequently eluted and separatedon a final, analytical C18 column before detection by themass spectrometer.

The Finnigan LTQ ion trap mass analyzer was set upto automatically run in MS and MS/MS mode. For eachscan, the five peptides generating the highest signal weresubjected to MS/MS to generate peptide fragmentationpatterns.

Results and Discussion

Data Analysis: MS/MS spectra obtained from the LC/MSanalysis were searched against a Swiss Prot database(release 7459) using the SEQUEST® algorithm (Eng et al,1994) implementation in BioWorks™ 3.1. Enzyme withtwo possible missed cleavages was specified as a searchparameter. The list of matched peptides was further evalu-ated using the Request/Unified scoring in BioWorks witha value of 2400 as the cut-off filter (Chelius et al, 2002).Additionally, the data was validated with a probability-based algorithm that calculated a statistical expectationvalue (SE-1) of database peptide matches based on theDeNovoX peptide sequencing pre-integration algorithm.(Pfeiffer et al, 2003). Peptides with a SE-1 > 10-5 wereaccepted for protein analysis. Post-translational modifica-tions were assessed using a fully-automated de novosequencing software program (DeNovoX™).

Figure 3: Plumbing Diagram for the MudPIT method on the Finnigan ProteomeX LTQ workstation. The method performs automated, on-linemultidimensional LC/MS. Peptides from a proteolytic digestion of a protein sample are loaded onto an SCX column, then eluted stepwise withsalt injections of increasing molarity. The eluted peptides are captured and desalted on small peptide traps and then eluted onto an analyticalcolumn for a final high resolution separation before detection by the Finnigan LTQ linear ion trap.

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Human cerebrospinal fluid is a complex fluid thatcontains various proteins ranging in concentration overat least 10 ten orders of magnitude. Using a combinationof multidimensional liquid chromatography and linear iontrap mass spectrometry, over 150 proteins were detectedwith high confidence from 400 µL of cerebrospinal fluidstarting material. More than 200,000 MS/MS sequencingattempts were made during the 2D separation of the

complex mixture. From this abundance of data weselected proteins that were identified by at least twoor more peptides for presentation in Tables 1 and 2.

Of the 150 proteins presented, 62 proteins werepreviously also identified in human plasma (Anderson etal, 2002) and are shown in Table 1. The other 92 proteinswere not described as plasma proteins previously andmight be specific to CSF (Table 2). Additional proteinsidentified in CSF by a single peptide are not shown here.

Accession Unique CoverageNumber Peptides [#] [%]P02763 ALPHA-1-ACID GLYCOPROTEIN 2 PRECURSOR 5 18P19652 ALPHA-1-ACID GLYCOPROTEIN 2 PRECURSOR 8 28P04217 ALPHA-1B-GLYCOPROTEIN 12 25P01009 Alpha-1-antitrypsin precursor 10 26P02765 ALPHA-2-HS-GLYCOPROTEIN PRECURSOR 10 31P01023 ALPHA-2-MACROGLOBULIN PRECURSOR 35 48P01011 ALPHA-1-ANTICHY MOTRYPSIN PRECURSOR 25 84P43652 AFAMIN PRECURSOR (ALPHA-ALBUMIN) 3 25P02768 SERUM ALBUMIN PRECURSOR 67 58P01876 IG ALPHA-1 CHAIN C REGION 2 25P01019 ANGIOTENSINOGEN PRECURSOR 11 25P01008 ANTITHROMBIN-III PRECURSOR (ATIII) 3 24P02647 APOLIPOPROTEIN A-I PRECURSOR (APR-AI) 6 16P02652 APOLIPOPROTEIN A-II PRECURSOR (APO-AII) 5 20P06727 APOLIPOPROTEIN A-IV PRECURSOR (APO-AIV) 4 15P06727 APOLIPOPROTEIN A-IV PRECURSOR (APO-AIV) 7 25P05090 APOLIPOPROTEIN D PRECURSOR 8 49P02649 APOLIPOPROTEIN E PRECURSOR (APO-E) 11 32P02749 Beta-2-glycoprotein I precursor (Apolipopro 5 12P32247 BOMBESIN RECEPTOR SUBTYPE-3 (BRS-3) 5 24P09871 COMPLEMENT C1S COMPONENT PRECURSOR 2 35P49454 CENP-F KINETOCHORE PROTEIN 3 45P00450 CERULOPLASMIN PRECURSOR (FERROXIDASE) 4 25P00751 COMPLEMENT FACTOR B PRECURSOR 23 37P08603 COMPLEMENT FACTOR H PRECURSOR 15 42P10909 Clusterin precursor (Complement-associated 15 25P09871 Complement C1s component precursor 2 24P01024 COMPLEMENT C3 PRECURSOR 38 37P01028 COMPLEMENT C4 PRECURSOR 32 23Q12860 CONTACTIN PRECURSOR (GLYCOPROTEIN GP135) 6 16P11532 DYSTROPHIN 5 25P02671 FIBRINOGEN ALPHA/ALPHA-E CHAIN PPRECURSOR 13 34P02751 FIBRONECTIN PRECURSOR (FN) (COLD-INSOLUBLE 3 24P01857 IG GAMMA-1 CHAIN C REGION 21 60P01859 IG GAMMA-2 CHAIN C REGION 14 30P01860 IG GAMMA-3 CHAIN C REGION (HEAVY CHAIN DISEA 13 34P01861 CHAIN C REGION 1 15P01777 HAIN V-III REGION TEI 1 14P01834 HAIN C REGION 3 50P80362 HAIN V-1 REGION WAT 7 21P18136 HAIN V-111 REGION PRECURSOR 6 16P06396 GELSOLIN PRECURSOR, PLASMA 16 27P02023 HEMOGLOBIN BETA CHAIN 32 23P02790 HEMOPEXIN PRECURSOR (BETA-1B-GLYCOPROTEIN) 12 25P00738 HAPTOGLOBIN-2 PRECURSOR 15 25P00739 HAPTOGLOBIN-RELATED PROTEIN PRECURSOR 2 24P01042 Kininogen Alpha-2-thiol proteinase inhibitor 38 37P01597 KAPPA CHAIN V-I REGION DEE 32 23P01842 IG LAMBDA CHAIN C REGIONS 3 50Q92876 KALLIKREIN 6 PRECURSOR (PROTEASE M) (NEURO 2 23P29622 Kallistatin precursor (Kallikrein inhibitor) 2 25Q9UM88 Beta 2-microglobulin protein 9 70P36955 MENT EPITHELIUM-DERIVED FACTOR PRECURSOR 6 23P05155 PLASMA PROTEASE C1 INHIBITOR PRECURSOR (C1 I 13 34P00747 PLASMINOGEN PRECURSOR 33 44P02787 SEROTRANSFERRIN PRECURSOR 34 44P05452 TETRANECTIN PRECURSOR (TN) (PPLASMINOGE-KRI 6 16P07477 TRYPSINOGEN I PRECURSOR 15 33P02766 Transthyretin precurosor (Prealbumin ) (TBPA) 6 16P02774 VITAMIN D0BINDING PROTEIN PRECURSOR (DBP) 7 18P04004 TRONECTIN PRECURSOR (SERUM SPREADING FACTOR) 2 35

Accession Protein1 P43652 Afamin precursor (Al;pha-albumin) (Alpha-Alb)2 P05091 ALDEHYDE DEHYDROGENASE, MITOCHONDRIAL PRECU3 P35221 Alpha-1 catenin (Dadherin-associated protein) (Alpha E-satenin)4 P05067 ALZHEIMER’S DISEASE AMYLOIC A4 PROTEIN PRECUR5 P51693 AMYLOID-LIKE PROTEIN 1 PRECURSOR (APLP)6 P20073 ANNEXIN A7 (ANNEXIN VII) (SYNEXIN)7 Q9BZC7 ATP-BINDING CASSETTE, SUB-FAMILY A, MEMBE8 P02730 Band 3 anion transport protein (Anion exchange protein 1)9 P01884 BETA-2-MICROGLOBULIN PRECURSOR10 P55290 CADHERN-13 PRECURSOR (TRUNCATED-CADHERN)11 O14967 Calmegin precursor12 P17655 Calpain 2, large[catalytic] subunit precursor (EC 3.4.22.17)13 Q15699 Cartilage homeoprotein 114 Q13033 Cell-cycle autoantigen SG2NA (S/G2 antigen)15 P54219 Chromaffin granule amine transporter (VAT1)16 P10645 CHROMOGRANIN A PRECURSOR (CGA) (PITUITARY17 O95239 CHROMOSOME-ASSOCIATED KINESIN DIF4A (CHRO18 Q9NYC9 CILIARY DY NEIN HEAVY CHAIN (AXONEMAL DYNE19 Q12860 Contactin precursor (Glycoprotein gP135)20 P01034 CYSTATIN C PRECURSOR (NEUROENDOCRINE BASIC21 P00156 CYTOCHROME B22 Q16850 CYTOCHROME P450 51 (CYPL1) (P450L1) (STERO23 P17611 Desmin24 Q9UBP4 DICKKOPF RELATED PROTEIN-3 PRECURSOR (DKK25 Q12805 EGF-CONTAINING FIBULIN-LIKE EXTRACELLULAR26 Q15668 EPIDIDYMAL SECRETORY PROTEIN E1 PRECURSOR27 P23142 FIBULIN-1 PRECURSOR28 Q9UEY8 GAMMA ADDUCIN (ADDUCIN-LIKE PROTEIN 70)29 Q9Y6H8 GAP JUNCTION ALPHA-3 PROTEIN (CONNEXIN 46)30 Q16478 Glutamate receptor, ionotropic kainite 5 precursor31 P55318 Hepatocyte nuclear factor 3-gamma (NF-3G)32 P18065 INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN33 P24592 Insulin-like growth factor binding protein34 Q9UKP45 Kelch-like protein 135 P10586 LAR protein precursor (Leukocyte antigen related)36 P03952 Lasma kallikrein precursor (EC 3.4.21.34)37 P08547 LINE-1 reverse transcriptase homolog38 P07864 L-lactate dehydrogenase C chain (LDH-C) (LD39 O94759 LONG TRANSIENT RECEPTOR POTENTIAL CHANNEL40 O00754 Lysosomal alpha-mannosidase precursor (Man41 P04156 MAJOR PRION PROTEIN PRECURSOR (PRP) (PRP27-42 P40925 Malate dehydrogenase, cytoplasmic (EC 1.1.1.37)43 O60476 Mannosyl-oligosaccharide 1,2-alpha-mannos44 P16035 Metalloproteinase inhibitor 2 precursor45 P20774 Mimecan precursor (Osteoglycin) (Osteoinductive factor)46 O95255 Multidrug resistance-associated protein 647 Q92859 Neogenin precursor48 P13591 NEURAL CELL ADHESION MOLECULE, 140 KDA ISO49 Q9N0X5 Neural proliferation differentiation and control protein-1 precursor50 P19022 Neural-cadherin precursor (N-cadherin) (Cadherin-2)51 P07197 Neurofilament triplet M protein (160 kDa neurofilament protein)52 O43613 Orexin receptor type 1 (Ox1r) (Hypocretin53 P20774 OSTEOINDUCTIVE FACTOR PRECURSOSR (OIF) (OSTEO54 P10451 OSTEOPONTIN PRECURSOR (BONE SIALOPROTEIN 1)55 P55058 PHOSPHOLIPID TRANSFER PROTEIN PRECURSOR (L56 P43034 PLATELET-ACTIVATING FACTOR ACETYLHYDROLASE57 P07602 Proactivator polypeptide precursor58 O60883 Probable rRNA processing protein EBP259 Q15113 Procollagen C-proteinase enhancer protein precursor (PCPE)60 P01210 PROENKEPHALIN A PRECURSOR [CONTAIN:MET-EN

Table 1: Typical blood serum proteins detected in CSF. Concentrationranges for proteins highlighted in blue are indicated in Figure 4.

Table 2: Proteins that were detected in CSF with at least two pep-tides, but are not described as typical blood plasma proteins(Anderson et al). Concentration ranges for proteins highlighted inblue are indicated in Figure 4.

Page 5 of 6

Figure 4 shows a log concentration plot for some ofthe proteins identified in this study, based upon consensusconcentration values reported elsewhere (J.N. Lycke et al,2003). The proteins presented in this figure are highlightedin blue in Tables 1 and 2.

Of particular significance is the detection and confi-dent (SE-1 >10-5) identification of several neurofilamentprotein M peptides. The neurofilament protein M is a

major structural protein of neurons and a marker foraxonal damage, and its concentration in CSF has previ-ously been reported to be below 125 ng/L in healthydonors. At a known amount of 100 µg total CSF proteinsinjected, the detection of neurofilament protein Mpeptides translates into a sub-femtomole detection sensi-tivity for the Data Dependent MS/MS analysis on a linearion trap mass spectrometer.

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Figure 4: Reference intervals for some plasma and CSF-specific proteins. Abundance of proteins is plotted on a log scale spanning 10 ordersof magnitude. Reference intervals for CSF proteins were obtained from J.N. Lycke et al 1998, R.M. Salomon et al 2003, G. Heine et al 2002.

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In addition to these

offices, Thermo Electron

Corporation maintains

a network of represen-

tative organizations

throughout the world.

Australia+61 2 9898 1244

Austria+43 1 333 50340

Belgium+32 2 482 30 30

Canada+1 800 532 4752

China+86 10 5850 3588

France+33 1 60 92 48 00

Germany+49 6103 4080

India+91 22 2778 1101

Italy+39 02 950 591

Japan+81 45 453 9100

Latin America+1 512 251 1503

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Conclusions• A total of 150 proteins were automatically detected in

CSF with high confidence using the Finnigan ProteomeXLTQ linear ion trap.

• Proteins were identified without the removal of highabundance proteins such as albumin and immuno-globulins.

• Typical CSF concentrations of the identified proteinsspan a range of seven orders of magnitude, with thelower end of this range in the tens of ng/L.

• The fast scan rates and the efficient tandem analysiscapabilities of the linear ion trap provide sub-fmol detec-tion sensitivity for the Data Dependent MS/MS analysis.

• The apparent detection limit of this technique is wellbelow that for proteins identified in gels and approachesthe range considered to represent the much-sought-afterlow abundance proteins.

References

Harrington, MG, Fonteh, AN, Stochaj, WA, Biringer, RG, Amato, H,Hühmer, AFR, Kilian, SC, Pogoda, JM, Chequer, RS, and Cowan, RP.A molecular catastrophe in migraine directs an understanding of patho-physiology and potential treatments. (submitted)

Andreasen, N, Minthon, L, Clarberg, A., Davisson, P, Gottfries, J,Vanmechelen, E, Vanderstichele, H, Winblad, B, Blennow, K. Sensitivity,specificity, and stability of CSF-tau in AD in a community-based patientsample. Neurology 1999; 53: 1488-1494.

Eng, J. K.; McCormack, A. L.; Yates, J. R. III. An approach to correlatetandem mass spectral data of peptides with amino acid sequences in aprotein database. J.Am.Soc.Mass Spectrom. 1994; 5: 976-989.

Chelius, D, Hühmer, AFR, Shieh, CH, Lehmberg, E, Traina, JA, Slattery, TK,and Erno Pungor, E Jr. Analysis of the Adenovirus Type 5 Proteome byLiquid Chromatography and Tandem Mass Spectrometry Methods.J. Proteome Res. 2002; 1:501-513.

Nehal T. Pfeiffer, Colette J. Rudd, Jim Shofstahl, Amy Zumwalt andFernando M. Maroto. Algorithm to Correlate MS/MS Spectra withTheoretical Peptide Fragmentation. Poster presentation, 51st ASMSConference on Mass Spectrometry, June 2003, Montreal, Quebec, Canada.

A.F. Hühmer, R.G. Biringer, H. Amato, M. Harrington. Measurement ofProtein Levels in Cerebrospinal Fluid Using Two Quantitation Strategies:ICAT-Labeling and Relative Peak Areas from Ion Chromatograms. Posterpresentation, ABRF Symposium, March 2003 Denver, CO, USA.

N.L. Anderson & N.G. Anderson. The Human Plasma Proteome Molecularand Cellular Proteomics 2002; 11:845-867

R.M. Salomon, B. Ripley, J.S. Kennedy, B. Johnson, D. Schmidt, J.M. Zeitzer,S.Nishino, E. Mignot. Diurnal Variation of Cerebrospinal Fluid Hypocretin-1(Orexin-A) Levels in Control and Depressed Subjects. Biol. Psychiatry 2003;54:96-104.

J.N. Lycke, J.E. Karlsson, O. Andersen, L.E. Rosengren. Neurofilament pro-tein in cerebrospinal fluid: a potential marker of activity in multiple sclerosis.J.Neurol.Neurosurg. Psychiatry 1998; 64:402-404.

G. Heine, H-D Zucht, M.U. Schuhmann, K. Bürger, Mürgens, M. Zumkeller,C.G. Schneekloth, H. Hampel, P.Schul-Knapper, H. Selle. High-resolution pep-tide mapping of cerebrospinal fluid: a novel concept for diagnosis andresearch in central nervous system diseases. J.Chromat. B 2002; 782:353-361.