Radically Improved Bottom-Up Protein Identificationusing a Finnigan LTQ Ion Trap Mass Spectrometer

ApplicationNote: 328

Key Words

• Sensitivity

• Quantitation

• Finnigan™ LTQ™

Chromatography and Mass SpectrometryApplication Note

Leo E. Bonilla, Rohan Thakur, and Andrew Guzzetta,Thermo Electron, San Jose, CA

Jacob D. Jaffe, Harvard Medical School, Boston, MA

IntroductionA key objective of proteomics projects is to developbiochemical and instrumental approaches to increasecoverage and confidence of protein identification. Recentadvances in mass spectrometry have placed this technologysolidly at the center of proteomics research. Ion traps, in

particular, are especially favored by most proteomicslaboratories due to their undisputed advantages in sensi-tivity, dynamic range, robustness, throughput, ease ofuse, and extremely favorable price-to-performance ratio.

The Finnigan LTQ, a new two-dimensional (2D)quadrupole (linear) ion trap (Figure 1), attains significantgains in performance over existing 3D and even othersimilar 2D-trapping devices. The technological andengineering advances present in the LTQ translate intoenhanced analytical throughput for proteomics analysisas measured by a larger number of high-confidenceprotein identifications obtained per analysis.

Figure 1. The Finnigan LTQ two-dimensional ion trap. The key design elements in this device are the axially segmented quadrupole assembly for efficienttrapping and mass analysis, and the dual detection system for maximum sensitivity.

GoalIn this report, we validate the enhanced performance ofthe LTQ in two key areas:

(1) The total number of protein identifications madeunder chromatographic conditions deliberatelydesigned to favor rapid co-elution of peptidespecies from complex tryptic digests.

(2) The confidence of protein identification asmeasured by higher protein consensus scores(SEQUEST™) and coverage, as well as by enhancedXcorr values obtained for peptide MS/MS spectra.

Experimental

Sample

To simplify data analysis, a well-defined and representa-tive set of tryptic peptides from Mycoplasma pneumoniaewas used. This organism was chosen for this study due tothe availability of its relatively small and well-annotatedgenome (689 predicted ORFs), which has no knowntranscriptional regulation.

Protein Isolation/ Digestion

Whole cell lysates of Mycoplasma pneumoniae wereprepared according to published protocols. All proteinswere digested together and the digest clarified. Off-linepeptide fractionation via strong cation exchange (SCX)chromatography (Whatman SCX, 4.6×250 mm) was per-formed, collecting 1 min fractions, which were transferredinto 96-well microtiter plates and dried by SpeedVac(Thermo Electron). All fractions were reconstituted into20 µL of 0.1% HCOOH just prior to LC/MS/MS analysis.

HPLC

Five SCX fractions (35-39, Figure 2) were selected foranalysis by nanospray ESI using the methods described inTable 1 below. The solvents used for LC/MS/MS analysiswere (A) 0.1% HCOOH and (B) 0.1% HCOOH in ACN.Automated capillary reversed-phase separations were per-formed on a Finnigan Surveyor™ MS binary pump andSurveyor autosampler (Thermo Electron, San Jose, CA)fitted with a pre-injector splitter (1:100) in order to gen-erate an effective column flow rate of about 250 nL/min.All MS analyses were done in parallel on both theFinnigan LCQ™ Deca XP Plus and the new Finnigan LTQlinear ion trap (Thermo Electron, San Jose, CA).

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20 40 60 80min

Fractions 35-39

1 mg lysate

mA

U

Finnigan LCQ Deca XP PlusLC SettingsColumn:

75 µm ID × 20 cm MAGIC C18 AQFlow rate: 250 nL/minEffective gradient time:

200 min (100 min)

MS SettingsData-dependent settings: full-scanMS (395-1800 m/z) plus top fiveMS/MS, default charge state +3,isolation width 3.0 m/z, normalizedcollision energy, 35%.Dynamic exclusion settings: repeatcount 3, repeat/exclusion duration30s/3 min, exclusion list size 50,exclusion mass with 3.0 m/z

Finnigan LTQLC SettingsColumn:

75 µm ID × 5 cm MAGIC C18 AQFlow rate: 250 nL/minEffective gradient time:

100 min

MS SettingsData-dependent settings: full-scanMS (395-1800 m/z) plus top fiveMS/MS, default charge state +3,isolation width 3.0 m/z, normalizedcollision energy, 35%.Dynamic exclusion settings: repeatcount 3, repeat/exclusion duration30s/3 min, exclusion list size 50,exclusion mass with 3.0 m/z

LC/MS Experimental Conditions

Table 1.Figure 2. Off-line pre-fractionation of tryptic peptides from Mycoplasmapneumoniae lysates. SCX was performed on a Whatman 4.6 × 250 mmcolumn and fractions collected at 1-min intervals.

Identical data-dependent MS/MS settings were usedfor data acquisition on both systems in order to facilitatethe comparison of results. In the case of the LTQ, onlya 100 min gradient was run using a 5-cm C18 column in order to deliberately challenge its performance underconditions that favor rapid co-elution of peptides (Figure3). Both 100 and 200 min gradients were run with theLCQ Deca XP Plus, using a 20 cm C18 column.

Database Search and AnalysisLC/MS/MS datasets were batch-searched usingBioWorks 3.1 (Thermo Electron, San Jose, CA) againstan indexed version of the Mycoplasma DB.(ftp://ftp.ncbi.nih.gov/genbank/genomes/Bacteria/Mycoplasma_pneumoniae/).

All spectra were searched assuming the following:(i) all tryptic cleavages, (ii) quantitative carboxyamido-methylation of cysteines, (iii) and differential oxidationof methionines. Charge state analysis of dta files was per-formed prior to searching and no PTMs were specified.The final SEQUEST output was filtered to display only

those sequences arising from peptide precursor ionsmeeting the following charge-state vs. Xcorr criteria: +1,Xcorr > 1.5; +2, Xcorr > 2.0; 3+, Xcorr > 2.5. Searchresults were compiled and evaluated using the multiplefile consensus tool of BioWorks.

For both the LCQ and LTQ experiments, the resultingnumber, coverage, spectral quality and confidence ofproteins identified were compared.

Results and DiscussionFigure 3 shows the reversed-phase capillary LC/MS/MSanalysis of fraction 37 on (A) the LCQ Deca XP Plusand (B) the LTQ. As discussed above, LC runs weretime-compressed in the case of the LTQ by using a shortercolumn and a shorter gradient time. This resulted in ahigher density of precursor ions being presented to theLTQ per unit time.

The average number of MS/MS scans produced perLC/MS/MS run in these experiments was approximately7,000 in (A) and 12,500 in (B).

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Figure 3. Reversed-phase capillary LC/MS/MS of fraction 37 on (A) the Finnigan LCQ Deca XP Plus and (B) the Finnigan LTQ ion traps.

A

B

number of searchable dta’s generated in these experi-ments was approximately 3,500 in the LCQ Deca XPPlus experiments and 15,000 in the LTQ experiments.

It is readily apparent that during the 100 min gradientexperiments, the LTQ identified approximately twice asmany proteins as the LCQ Deca XP Plus (548 vs. 253);this in spite of the fact that the LTQ was fitted with ashorter (5 cm) column than the LCQ Deca XP Plus(20 cm). As the gradient time was extended to 200 minfor the LCQ Deca XP Plus, while keeping it at 100 minfor the LTQ, both instruments were able to make asimilar number of proteins IDs (575 vs. 548). Theseobservations provide evidence for the superiorthroughput capabilities of the new Finnigan LTQ.

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Figure 4 shows a comparison of single (unaveraged)MS/MS spectra and their corresponding SEQUEST Xcorrvalues. These spectra represent two sets of identicaltryptic peptides acquired in data-dependent mode duringLC/MS/MS analysis on (A) the LCQ Deca XP Plus (leftpanel) and (B) the LTQ (right panel). Notice the signifi-cant qualitative difference between LCQ and LTQspectra. The LTQ spectra display noticeably higher S/Nwhich, in turn, translates into more y- and b- ionsmatched during database searches. Quantitatively, thisis reflected by higher Xcorr values, yielding increasedconfidence in protein identifications.

Figure 5 shows the total combined number of proteinsidentified in fractions 35-39. In each case, the numberof protein IDs was determined by simply filtering theSEQUEST output according to the charge state vs. Xcorrsettings described in the experimental section above.Manual verification of IDs was not attempted. The

A B

Figure 4. Comparison of single MS/MS spectra and their corresponding SEQUEST™ Xcorr values for identical tryptic peptides acquired during data-dependentLC/MS/MS analysis on (A) the Finnigan LCQ Deca XP Plus and (B) Finnigan LTQ ion traps. Notice the significant difference in spectral quality and Xcorr valuesfor the data obtained from the LTQ.

Figure 6 compares percent sequence coverage (byamino acid number) for the top-ten proteins identifiedin fractions 35-39 using the three different experimentalsetups described. Under identical gradient conditions (i.e.100 min), the LTQ outperforms the LCQ Deca XP Plusin 9 out of 10 cases.

BioWorks protein Consensus Score combines all of theinformation from all of the peptides used to identify aprotein into a single value, which can be used to assessthe overall quality of the identification. Figure 7 showsa comparison of the consensus scores for the top-tenproteins identified in fractions 35-39. Here again, it isclear that the data obtained from the LTQ experimentsresult in significantly higher protein scores which, in turn,translate into better, more confident protein IDs.

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Figure 5. Total number of protein IDs in fractions 35-39.

Nth Rank Protein Name

1 Leucine Aminopeptidase2 Pyruvate dehydrogenase E1 beta-subunit3 Molecular chaperone4 DNA-directed RNA polymerase beta chain5 RNA polymerase beta subunit6 Ribosomal protein L17 Conserved hypothetical protein8 PEP-dependent HPr protein kinase phosphoryltransferase9 L-lactate dehydrogenase

10 Heat-shock protein GroEL

Figure 6. Comparison of percent sequence coverage (by AAs) for the top-ten IDs in fractions 35-39.

Conclusions The Finnigan LTQ offers clear performance advantagesover other existing MS technologies. Its key advantagesfor proteomics experiments are:

1) Considerably faster overall scan cycle, resulting inthe acquisition of time-compressed, richer MS/MSdatasets. These datasets represent a larger numberof MS/MS sequencing attempts per unit time.Therefore, it is not surprising that the FinniganLTQ produces a higher number of peptide/proteinIDs, with higher overall percent sequence coverage,all in a fraction of the time required by theFinnigan LCQ Deca XP Plus.

2) The ability to ‘get to baseline’ fast through effi-cient implementation of data-dependent analysesand dynamic exclusion. A unique advantage of amore sensitive, faster scanning LTQ is its enhancedability to detect and acquire MS/MS data on lowintensity precursor ions. Routine proteomicsanalyses on the LTQ are being performed usingsettings for data-dependent MS/MS analyses onthe top-10 precursor ions with a repeat count of 1(data not shown). This is a critical capability inorder to be able to detect rare, low-copy numberproteins, as well as to increase protein sequencecoverage.

3) The remarkable spectral quality of LTQ datayields consistently higher Xcorr and Consensusvalues. This has a direct and positive effect on theconfidence of protein IDs stemming from databasesearches. Furthermore, it has also been demon-strated to enhance the performance of probability-based de novo sequencing algorithms such asDeNovoX™ (data not shown).

In summary, the advanced capabilities of the LTQ arealready redefining the current state-of-the-art of pro-teomics capabilities worldwide. By effectively extendingthe practical dynamic range of the typical proteomicsexperiment, the LTQ has become a vital tool to attacktough biological questions such as the discovery anddetection of low-abundance proteins and identificationof disease markers in complex matrices, like serumor plasma, where the dynamic range of proteinconcentrations is 1012.

©2003 Thermo Electron Corporation. All rights reserved. SEQUEST is a trademark of the University of Washington. All other trade-marks are the property of Thermo Electron Corporation and its subsidiaries. Specifications, terms and pricing are subject to change.Not all products are available in all countries. Please consult your local sales representative for details. Printed in the USA.

Nth Rank Protein Name

1 Leucine Aminopeptidase2 Pyruvate dehydrogenase E1 beta-subunit3 Molecular chaperone4 DNA-directed RNA polymerase beta chain5 RNA polymerase beta subunit6 Ribosomal protein L17 Conserved hypothetical protein8 PEP-dependent HPr protein kinase phosphoryltransferase9 L-lactate dehydrogenase

10 Heat-shock protein GroEL

Figure 7. Comparison of SEQUEST consensus scores for the top-ten protein IDs in fractions 35-39.

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