Overcoming Undersampling in Proteomic Experiments with the Help of a Novel Linear Trap-Orbitrap Mass Spectrometer Eugen Damoc 1 , Eduard Denisov 1 , Alexander Makarov 1 , Justin Blethrow 2 , Tonya Pekar Second 2 , Vlad Zabrouskov 2 , Jens Griep-Raming 1 , Thomas Moehring 1 1 Thermo Fisher Scientific, Bremen, Germany, 2 Thermo Fisher Scientific, San Jose, CA, USA Conclusions  With more than twice faster MS/MS scan speed an LTQ Orbitrap XL, the new hybrid instrument helps overcoming undersampling, increasing the depth of analysis of complex proteomes.  The enhanced sensitivity and improved duty cycle of the new instrument enables a dramatic increase of the number of unique peptide IDs (see Figure. 12), which translates into an increased number of proteins identified with higher sequence coverage and confidence. Overview Purpose: Evaluate the performance of the newly developed hybrid linear ion trap-Orbitrap mass spectrometer for characterization of complex proteomes Methods: NanoLC-MS/MS analyses of complex protein digests (C. elegans and E. coli digests) Results: The increased sensitivity and improved duty cycle of the new instrument enables a dramatic increase in the number of protein IDs with increased sequence coverage and high confidence. Introduction Enormous improvements in mass spectrometry technology over the last few years have allowed for ever deepening analysis of complex proteomes. However, many low- abundance proteins remain undetected in current large-scale proteomic analyses due to the limits on the rate at which MS/MS spectra can be acquired. To solve this undersampling problem, we developed a new hybrid linear ion trap-Orbitrap mass spectrometer (LTQ Orbitrap Velos, Figure 1) that provides faster MS/MS scanning in the ion trap (up to 10 peptide sequences per second), thereby increasing the depth of analysis of complex protein mixtures. In the present work, we evaluated the ability of the newly developed hybrid instrument to identify more proteins, with increased sequence coverage and confidence. Methods Significant technology improvements have been implemented in the Thermo Scientific LTQ Orbitrap Velos, including: (a) a progressive stacked-ring ion guide (S-Lens) with 5- 10x increased ion transmission 1 ; (b) a dual-pressure differentially pumped ion trap with a higher-pressure cell for improved ion trapping, isolation, and CID efficiencies, and a lower- pressure cell for improved resolution and/or scan speed 2 ; (c) predictive AGC for increased scan speed 3 ; (d) an HCD-collision cell with axial field for improved performance; (e) improved vacuum in the Orbitrap TM chamber for better intact protein analysis. To assess improvements in proteome coverage, nanoLC-MS/MS analyses of C. elegans and E. coli protein digests were performed using various LC gradients. These two complex peptide mixtures were separated using a Thermo Scientific Surveyor LC and MicroAS autosampler with a reversed-phase peptide trap (100 μm inner diameter, 2 cm length) and a reversed-phase analytical column (75 μm inner diameter, 10 cm length, 3 μm particle size, both Nanoseparations, NL), at a flow rate of 300 nL/min. The raw data files from duplicate analyses were searched with Mascot using Thermo Scientific Proteome Discoverer software Results NanoLC-MS/MS analysis of 1 μg C. elegans protein digest was carried out using two different LC gradients (0-40% acetonitrile in 75 minutes, and 0-40% acetonitrile in 150 minutes). Using the new instrument, a two-fold increase in the number of MS/MS spectra acquired per unit time was observed. This provided identification with the LTQ Orbitrap Velos TM of a much higher number of proteins and peptides in the 75-minute run than in the 150-minute run using A Thermo Scientific LTQ Orbitrap XL instrument (see Figure 3). FIGURE 2. NanoLC-MS/MS analysis of 1 μg C. elegans digest using TOP20 ITMS2 method (8-9Hz for ITMS2 CID scans, 5e3 ions) FIGURE 4. Proteome Discoverer TM views showing the increase in sequence coverage by using the LTQ Orbitrap Velos FIGURE 5. NanoLC-MS/MS analysis of 100 ng E. coli digest using TOP20 ITMS2 method (1x OT full scan (R=100k) + 20x ddITMS2 CID scans) FIGURE 8. NanoLC MS/MS analysis of 100 ng E. coli digest using TOP10 HCD method (1x OT full scan (R=30k) + 10x ddHCD scans) References 1. E. R. Wouters, M. Splendore, M.W. Senko, J. E. P. Syka, J.-J. Dunyach, “Design of a progressively spaced stacked ring ion guide for improved ion transmission at high pressure”, ASMS 2008. 2. J. C. Schwartz, J. E. P. Syka, P. M. Remes, S. T. Quarmby, “A New Dual Cell Linear Ion Trap Configuration for Improved Quadrupole Ion Trap Performance”, ASMS 2008. 3. B. Brazdova, J. Horner, J. Phillips, “Improved Coverage in Global Proteomics Survey Experiments by Decreasing Cycle Time on a Linear Ion Trap Mass Spectrometer, ASMS 2008. Mascot is a trademark of Matrix Science. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. C:\Xcalibur\...\1ug_CE_short_CID_top20_1 4/24/2009 3:03:23 PM RT: 19.28149 - 75.48315 20 25 30 35 40 45 50 55 60 65 70 75 Time (min) 0 20 40 60 80 100 Relative Abundance 41.39383 652.36017 32.00259 566.76666 41.30246 652.36090 41.48560 652.36017 32.04841 566.76678 31.96205 566.76642 52.28502 788.91937 41.26801 652.36090 41.52585 652.36060 46.22037 889.44312 52.38579 788.91833 41.57211 652.36060 29.74806 506.23923 56.52121 601.34619 29.30598 452.92972 56.87537 687.86023 64.20935 567.82465 25.96189 458.77594 67.48466 607.06696 70.19599 743.41492 NL: 1.53E8 Base Peak MS 1ug_CE_sh ort_CID_top 20_1 RT: 31.73600 - 31.91753 31.74 31.76 31.78 31.80 31.82 31.84 31.86 31.88 31.90 Time (min) 0 10 20 30 40 50 60 70 Relative Abundance 31.89323 457.78662 31.80148 504.30307 31.84701 457.78699 31.75487 504.30316 31.90046 557.94135 31.85438 788.74042 31.81069 448.95166 31.76226 447.81412 31.86039 602.43695 31.73898 512.55280 31.84070 481.26996 31.90782 720.08545 31.81770 869.28290 31.88740 584.05280 31.86942 539.96014 31.78385 572.50055 31.79755 516.19843 NL: 3.26E7 Base Peak MS 1ug_CE_sh ort_CID_top 20_1 1ug_CE_short_CID_top20_1#5005 RT: 31.85 AV: 1 NL: 1.74E7 T: FTMS + p NSI Full ms [350.00-1800.00] 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z 0 20 40 60 80 100 Relative Abundance 457.78699 R=62501 z=2 504.30313 R=60201 z=2 617.97437 R=53901 z=3 441.22952 R=63601 z=2 767.38660 R=48701 z=2 523.26398 R=59801 z=3 660.34827 R=51900 z=3 926.96039 R=44100 z=2 818.02960 R=45800 z=3 990.02020 R=43600 z=2 1085.52246 R=38400 z=2 1255.26416 R=37400 z=3 1191.60168 R=38201 z=1 1346.56433 R=28204 z=? 2.79s 2.73s 2.77s TOP20 CID TOP20 CID TOP20 CID FTMS, 1e6, R=60,000 Base Peak Chromatogram C:\Xcalibur\...\1ug_CE_short_CID_top20_1 4/24/2009 3:03:23 PM RT: 19.28149 - 75.48315 20 25 30 35 40 45 50 55 60 65 70 75 Time (min) 0 20 40 60 80 100 Relative Abundance 41.39383 652.36017 32.00259 566.76666 41.30246 652.36090 41.48560 652.36017 32.04841 566.76678 31.96205 566.76642 52.28502 788.91937 41.26801 652.36090 41.52585 652.36060 46.22037 889.44312 52.38579 788.91833 41.57211 652.36060 29.74806 506.23923 56.52121 601.34619 29.30598 452.92972 56.87537 687.86023 64.20935 567.82465 25.96189 458.77594 67.48466 607.06696 70.19599 743.41492 NL: 1.53E8 Base Peak MS 1ug_CE_sh ort_CID_top 20_1 RT: 31.73600 - 31.91753 31.74 31.76 31.78 31.80 31.82 31.84 31.86 31.88 31.90 Time (min) 0 10 20 30 40 50 60 70 Relative Abundance 31.89323 457.78662 31.80148 504.30307 31.84701 457.78699 31.75487 504.30316 31.90046 557.94135 31.85438 788.74042 31.81069 448.95166 31.76226 447.81412 31.86039 602.43695 31.73898 512.55280 31.84070 481.26996 31.90782 720.08545 31.81770 869.28290 31.88740 584.05280 31.86942 539.96014 31.78385 572.50055 31.79755 516.19843 NL: 3.26E7 Base Peak MS 1ug_CE_sh ort_CID_top 20_1 1ug_CE_short_CID_top20_1#5005 RT: 31.85 AV: 1 NL: 1.74E7 T: FTMS + p NSI Full ms [350.00-1800.00] 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z 0 20 40 60 80 100 Relative Abundance 457.78699 R=62501 z=2 504.30313 R=60201 z=2 617.97437 R=53901 z=3 441.22952 R=63601 z=2 767.38660 R=48701 z=2 523.26398 R=59801 z=3 660.34827 R=51900 z=3 926.96039 R=44100 z=2 818.02960 R=45800 z=3 990.02020 R=43600 z=2 1085.52246 R=38400 z=2 1255.26416 R=37400 z=3 1191.60168 R=38201 z=1 1346.56433 R=28204 z=? C:\Xcalibur\...\1ug_CE_short_CID_top20_1 4/24/2009 3:03:23 PM RT: 19.28149 - 75.48315 20 25 30 35 40 45 50 55 60 65 70 75 Time (min) 0 20 40 60 80 100 Relative Abundance C:\Xcalibur\...\1ug_CE_short_CID_top20_1 4/24/2009 3:03:23 PM RT: 19.28149 - 75.48315 20 25 30 35 40 45 50 55 60 65 70 75 Time (min) 0 20 40 60 80 100 Relative Abundance 41.39383 652.36017 32.00259 566.76666 41.30246 652.36090 41.48560 652.36017 32.04841 566.76678 31.96205 566.76642 52.28502 788.91937 41.26801 652.36090 41.52585 652.36060 46.22037 889.44312 52.38579 788.91833 41.57211 652.36060 29.74806 506.23923 56.52121 601.34619 29.30598 452.92972 56.87537 687.86023 64.20935 567.82465 25.96189 458.77594 67.48466 607.06696 70.19599 743.41492 NL: 1.53E8 Base Peak MS 1ug_CE_sh ort_CID_top 20_1 RT: 31.73600 - 31.91753 41.39383 652.36017 32.00259 566.76666 41.30246 652.36090 41.48560 652.36017 32.04841 566.76678 31.96205 566.76642 52.28502 788.91937 41.26801 652.36090 41.52585 652.36060 46.22037 889.44312 52.38579 788.91833 41.57211 652.36060 29.74806 506.23923 56.52121 601.34619 29.30598 452.92972 56.87537 687.86023 64.20935 567.82465 25.96189 458.77594 67.48466 607.06696 70.19599 743.41492 NL: 1.53E8 Base Peak MS 1ug_CE_sh ort_CID_top 20_1 RT: 31.73600 - 31.91753 31.74 31.76 31.78 31.80 31.82 31.84 31.86 31.88 31.90 Time (min) 0 10 20 30 40 50 60 70 Relative Abundance 31.89323 457.78662 31.80148 504.30307 31.84701 457.78699 31.75487 504.30316 31.90046 557.94135 31.85438 788.74042 31.81069 448.95166 31.76226 447.81412 31.86039 602.43695 31.73898 512.55280 31.84070 481.26996 31.90782 720.08545 31.81770 869.28290 31.88740 584.05280 31.86942 539.96014 31.78385 572.50055 31.79755 516.19843 NL: 3.26E7 Base Peak MS 1ug_CE_sh ort_CID_top 20_1 1ug_CE_short_CID_top20_1#5005 RT: 31.85 AV: 1 NL: 1.74E7 T: FTMS + p NSI Full ms [350.00-1800.00] 31.74 31.76 31.78 31.80 31.82 31.84 31.86 31.88 31.90 Time (min) 0 10 20 30 40 50 60 70 Relative Abundance 31.89323 457.78662 31.80148 504.30307 31.84701 457.78699 31.75487 504.30316 31.90046 557.94135 31.85438 788.74042 31.81069 448.95166 31.76226 447.81412 31.86039 602.43695 31.73898 512.55280 31.84070 481.26996 31.90782 720.08545 31.81770 869.28290 31.88740 584.05280 31.86942 539.96014 31.78385 572.50055 31.79755 516.19843 NL: 3.26E7 Base Peak MS 1ug_CE_sh ort_CID_top 20_1 1ug_CE_short_CID_top20_1#5005 RT: 31.85 AV: 1 NL: 1.74E7 T: FTMS + p NSI Full ms [350.00-1800.00] 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z 0 20 40 60 80 100 Relative Abundance 457.78699 R=62501 z=2 504.30313 R=60201 z=2 617.97437 R=53901 z=3 441.22952 R=63601 z=2 767.38660 R=48701 z=2 523.26398 R=59801 z=3 660.34827 R=51900 z=3 926.96039 R=44100 z=2 818.02960 R=45800 z=3 990.02020 R=43600 z=2 1085.52246 R=38400 z=2 1255.26416 R=37400 z=3 1191.60168 R=38201 z=1 1346.56433 R=28204 z=? 2.79s 2.73s 2.77s TOP20 CID TOP20 CID TOP20 CID FTMS, 1e6, R=60,000 Base Peak Chromatogram FIGURE 3. Proteins and unique peptides identified at <1% FDR in 1μg C. Elegans digest with LTQ Orbitrap XL and LTQ Orbitrap Velos using two different LC gradients (0-40% ACN in 75 min and 0-40% ACN in 150 min) Protein IDs at <1% FDR 0 200 400 600 800 1000 1200 1400 75' 150' 75' 150' LTQ OT XL LTQ OT Velos 609 762 1049 1253 64.44% 72.25% 0 200 400 600 800 1000 1200 1400 75' 150' 75' 150' LTQ OT XL LTQ OT Velos 609 762 1049 1253 64.44% 72.25% 0 500 1000 1500 2000 2500 3000 3500 4000 4500 75’ 150’ 75’ 150’ LTQ OT XL LTQ OT Velos 74.32% 83.83% 1750 2418 3217 4215 Unique Peptide IDs at <1% FDR 0 500 1000 1500 2000 2500 3000 3500 4000 4500 75’ 150’ 75’ 150’ LTQ OT XL LTQ OT Velos 74.32% 83.83% 1750 2418 3217 4215 Unique Peptide IDs at <1% FDR ecoli_redalk_115minrun_90mingrad_top1... 5/10/2009 6:53:55 PM RT: 0.00000 - 114.99123 0 10 20 30 40 50 60 70 80 90 100 110 Time (min) 0 20 40 60 80 100 Relative Abundance 53.92451 601.96735 43.39184 688.82166 87.85834 594.31989 59.60684 514.29828 102.46327 352.33865 26.08003 442.94952 43.27784 688.82178 90.00384 652.39667 102.65961 352.33859 59.78117 514.29803 33.79367 388.70734 103.23141 352.33871 101.86078 352.33902 87.74901 594.32147 24.11667 449.74420 62.31217 512.64685 68.65836 582.28821 39.06450 672.82825 1.70606 360.14914 103.26726 352.33881 101.50205 473.95746 15.64318 377.21536 71.69317 630.33618 1.82236 365.10492 105.93670 391.28323 NL: 7.64E6 Base Peak MS ecoli_redalk_115m inrun_90mingrad_t op10_HCD_SF10 0_100ng_2 RT: 60.97285 - 61.05496 60.98 60.99 61.00 61.01 61.02 61.03 61.04 61.05 Time (min) 0 5 10 15 20 25 30 35 40 Relative Abundance 60.98768 625.30811 61.04651 625.30786 61.00693 458.71072 61.02067 359.20169 60.98018 448.25299 61.02525 413.26407 61.00241 719.36084 60.99869 1187.64319 61.01146 881.43304 60.97556 541.11737 61.03893 906.42645 61.01606 545.30042 61.03433 1358.67175 NL: 4.64E5 Base Peak MS ecoli_redalk_115m inrun_90mingrad_t op10_HCD_SF10 0_100ng_2 ecoli_redalk_115minrun_90mingrad_top10_HCD_SF100_100ng_2#9961 RT: 60.99 AV: 1 NL: 4.62E5 T: FTMS + p NSI Full ms [350.00-1800.00] 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 m/z 0 20 40 60 80 100 Relative Abundance 625.30804 R=27100 z=4 500.44780 R=30000 z=5 552.26456 R=28700 z=4 652.57560 R=26200 z=4 736.01684 R=25100 z=3 429.08817 R=32301 z=1 391.28371 R=33701 z=1 842.49838 R=23201 z=1 715.81115 R=24801 z=2 775.02876 R=24200 z=3 457.57318 R=31801 z=3 1103.52439 R=20100 z=2 986.85729 R=21400 z=3 916.73703 R=21400 z=3 1159.88462 R=10104 z=? 1046.00169 R=18700 z=1 3.53s 1x OT full scan (R=30k) + 10x ddHCD scans 1 2 3 4 5 6 7 8 9 10 FTMS, 1e6, R=30,000 Base Peak Chromatogram ecoli_redalk_115minrun_90mingrad_top1... 5/10/2009 6:53:55 PM RT: 0.00000 - 114.99123 0 10 20 30 40 50 60 70 80 90 100 110 Time (min) 0 20 40 60 80 100 Relative Abundance 53.92451 601.96735 43.39184 688.82166 87.85834 594.31989 59.60684 514.29828 102.46327 352.33865 26.08003 442.94952 43.27784 688.82178 90.00384 652.39667 102.65961 352.33859 59.78117 514.29803 33.79367 388.70734 103.23141 352.33871 101.86078 352.33902 87.74901 594.32147 24.11667 449.74420 62.31217 512.64685 68.65836 582.28821 39.06450 672.82825 1.70606 360.14914 103.26726 352.33881 101.50205 473.95746 15.64318 377.21536 71.69317 630.33618 1.82236 365.10492 105.93670 391.28323 NL: 7.64E6 Base Peak MS ecoli_redalk_115m inrun_90mingrad_t op10_HCD_SF10 0_100ng_2 RT: 60.97285 - 61.05496 60.98 60.99 61.00 61.01 61.02 61.03 61.04 61.05 Time (min) 0 5 10 15 20 25 30 35 40 Relative Abundance 60.98768 625.30811 61.04651 625.30786 61.00693 458.71072 61.02067 359.20169 60.98018 448.25299 61.02525 413.26407 61.00241 719.36084 60.99869 1187.64319 61.01146 881.43304 60.97556 541.11737 61.03893 906.42645 61.01606 545.30042 61.03433 1358.67175 NL: 4.64E5 Base Peak MS ecoli_redalk_115m inrun_90mingrad_t op10_HCD_SF10 0_100ng_2 ecoli_redalk_115minrun_90mingrad_top10_HCD_SF100_100ng_2#9961 RT: 60.99 AV: 1 NL: 4.62E5 T: FTMS + p NSI Full ms [350.00-1800.00] 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 m/z 0 20 40 60 80 100 Relative Abundance 625.30804 R=27100 z=4 500.44780 R=30000 z=5 552.26456 R=28700 z=4 652.57560 R=26200 z=4 736.01684 R=25100 z=3 429.08817 R=32301 z=1 391.28371 R=33701 z=1 842.49838 R=23201 z=1 715.81115 R=24801 z=2 775.02876 R=24200 z=3 457.57318 R=31801 z=3 1103.52439 R=20100 z=2 986.85729 R=21400 z=3 916.73703 R=21400 z=3 1159.88462 R=10104 z=? 1046.00169 R=18700 z=1 ecoli_redalk_115minrun_90mingrad_top1... 5/10/2009 6:53:55 PM RT: 0.00000 - 114.99123 0 10 20 30 40 50 60 70 80 90 100 110 Time (min) 0 20 40 60 80 100 Relative Abundance ecoli_redalk_115minrun_90mingrad_top1... 5/10/2009 6:53:55 PM RT: 0.00000 - 114.99123 0 10 20 30 40 50 60 70 80 90 100 110 Time (min) 0 20 40 60 80 100 Relative Abundance 53.92451 601.96735 43.39184 688.82166 87.85834 594.31989 59.60684 514.29828 102.46327 352.33865 26.08003 442.94952 43.27784 688.82178 90.00384 652.39667 102.65961 352.33859 59.78117 514.29803 33.79367 388.70734 103.23141 352.33871 101.86078 352.33902 87.74901 594.32147 24.11667 449.74420 62.31217 512.64685 68.65836 582.28821 39.06450 672.82825 1.70606 360.14914 103.26726 352.33881 101.50205 473.95746 15.64318 377.21536 71.69317 630.33618 1.82236 365.10492 105.93670 391.28323 NL: 7.64E6 Base Peak MS ecoli_redalk_115m inrun_90mingrad_t op10_HCD_SF10 0_100ng_2 RT: 60.97285 - 61.05496 53.92451 601.96735 43.39184 688.82166 87.85834 594.31989 59.60684 514.29828 102.46327 352.33865 26.08003 442.94952 43.27784 688.82178 90.00384 652.39667 102.65961 352.33859 59.78117 514.29803 33.79367 388.70734 103.23141 352.33871 101.86078 352.33902 87.74901 594.32147 24.11667 449.74420 62.31217 512.64685 68.65836 582.28821 39.06450 672.82825 1.70606 360.14914 103.26726 352.33881 101.50205 473.95746 15.64318 377.21536 71.69317 630.33618 1.82236 365.10492 105.93670 391.28323 NL: 7.64E6 Base Peak MS ecoli_redalk_115m inrun_90mingrad_t op10_HCD_SF10 0_100ng_2 RT: 60.97285 - 61.05496 60.98 60.99 61.00 61.01 61.02 61.03 61.04 61.05 Time (min) 0 5 10 15 20 25 30 35 40 Relative Abundance 60.98768 625.30811 61.04651 625.30786 61.00693 458.71072 61.02067 359.20169 60.98018 448.25299 61.02525 413.26407 61.00241 719.36084 60.99869 1187.64319 61.01146 881.43304 60.97556 541.11737 61.03893 906.42645 61.01606 545.30042 61.03433 1358.67175 NL: 4.64E5 Base Peak MS ecoli_redalk_115m inrun_90mingrad_t op10_HCD_SF10 0_100ng_2 ecoli_redalk_115minrun_90mingrad_top10_HCD_SF100_100ng_2#9961 RT: 60.99 AV: 1 NL: 4.62E5 T: FTMS + p NSI Full ms [350.00-1800.00] 60.98 60.99 61.00 61.01 61.02 61.03 61.04 61.05 Time (min) 0 5 10 15 20 25 30 35 40 Relative Abundance 60.98768 625.30811 61.04651 625.30786 61.00693 458.71072 61.02067 359.20169 60.98018 448.25299 61.02525 413.26407 61.00241 719.36084 60.99869 1187.64319 61.01146 881.43304 60.97556 541.11737 61.03893 906.42645 61.01606 545.30042 61.03433 1358.67175 NL: 4.64E5 Base Peak MS ecoli_redalk_115m inrun_90mingrad_t op10_HCD_SF10 0_100ng_2 ecoli_redalk_115minrun_90mingrad_top10_HCD_SF100_100ng_2#9961 RT: 60.99 AV: 1 NL: 4.62E5 T: FTMS + p NSI Full ms [350.00-1800.00] 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 m/z 0 20 40 60 80 100 Relative Abundance 625.30804 R=27100 z=4 500.44780 R=30000 z=5 552.26456 R=28700 z=4 652.57560 R=26200 z=4 736.01684 R=25100 z=3 429.08817 R=32301 z=1 391.28371 R=33701 z=1 842.49838 R=23201 z=1 715.81115 R=24801 z=2 775.02876 R=24200 z=3 457.57318 R=31801 z=3 1103.52439 R=20100 z=2 986.85729 R=21400 z=3 916.73703 R=21400 z=3 1159.88462 R=10104 z=? 1046.00169 R=18700 z=1 3.53s 1x OT full scan (R=30k) + 10x ddHCD scans 1 2 3 4 5 6 7 8 9 10 FTMS, 1e6, R=30,000 Base Peak Chromatogram FIGURE 9. Identification of 10 peptides from 10 consecutive HCD spectra 1 2 3 4 5 6 7 8 9 10 ecoli_redalk_115minrun_90mingrad_top1... 2/25/2009 9:32:13 PM ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9962 RT:61.00 AV: 1 NL: 1.99E4 T: FTMS + p NSI d Full ms2 758.42@hcd35.00 [100.00-1530.00] 200 400 600 800 1000 1200 1400 m/z 0 20 40 60 80 100 Relative Abundance 1187.64832 z=1 200.13951 z=? 594.32690 z=2 806.44598 z=1 329.18198 z=1 905.51459 z=1 1288.69727 z=1 644.84998 z=2 1090.59802 z=1 382.20880 z=? 511.25323 z=2 1467.69104 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9963 RT: 61.00 AV:1 NL:2.39E4 T: FTMS + p NSI d Full ms2 579.95@hcd35.00 [100.00-1750.00] 200 400 600 800 1000 1200 1400 1600 m/z 0 20 40 60 80 100 Relative Abundance 719.36469 z=2 589.28918 z=2 812.39856 z=2 522.61646 z=3 301.11505 z=1 866.43231 z=2 468.22293 z=2 1177.57947 z=? 1306.05542 z=? 1552.11975 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9964 RT:61.01 AV: 1 NL: 7.80E4 T: FTMS + p NSI d Full ms2 431.47@hcd35.00 [100.00-1740.00] 200 400 600 800 1000 1200 1400 1600 m/z 0 20 40 60 80 100 Relative Abundance 458.71375 z=2 581.78229 z=2 916.42065 z=1 365.19400 z=1 219.14963 z=? 815.37323 z=1 708.38354 z=1 1015.49182 z=1 1453.09192 z=? 1312.03992 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9965 RT: 61.01 AV:1 NL:1.38E4 T: FTMS + p NSI d Full ms2 727.37@hcd35.00 [100.00-1465.00] 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z 0 20 40 60 80 100 Relative Abundance 881.43732 z=1 727.36548 z=2 573.28967 z=1 487.17896 z=1 129.10245 z=? 600.26361 z=1 359.15573 z=? 967.55438 z=? 1054.58350 z=? 854.45422 z=? 1326.62781 z=1 ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9966 RT:61.02 AV: 1 NL: 1.32E4 T: FTMS + p NSI d Full ms2 558.80@hcd35.00 [100.00-1130.00] 100 200 300 400 500 600 700 800 900 1000 1100 m/z 0 20 40 60 80 100 Relative Abundance 545.30371 z=1 215.13892 z=1 874.46344 z=1 745.41852 z=1 432.22049 z=1 688.39899 z=1 816.45746 z=1 303.17639 z=? 243.13406 z=? 173.12830 z=? 915.53046 z=1 516.31775 z=1 1016.56140 z=? 615.34186 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9967 RT: 61.02 AV:1 NL:7.68E4 T: FTMS + p NSI d Full ms2 406.57@hcd35.00 [100.00-1230.00] 100 200 300 400 500 600 700 800 900 1000 1100 1200 m/z 0 20 40 60 80 100 Relative Abundance 359.20441 z=? 746.42029 z=1 647.35168 z=1 472.28802 z=1 180.10544 z=2 407.23026 z=? 718.42682 z=1 310.18143 z=2 859.50500 z=1 571.36005 z=? 931.03668 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9968 RT:61.03 AV: 1 NL: 3.96E4 T: FTMS + p NSI d Full ms2 411.91@hcd35.00 [100.00-1250.00] 100 200 300 400 500 600 700 800 900 1000 1100 1200 m/z 0 20 40 60 80 100 Relative Abundance 413.26700 z=? 545.34497 z=1 360.19949 z=1 658.42993 z=1 289.16245 z=? 217.08179 z=? 874.50885 z=? 759.47412 z=? 967.67554 z=? 1145.65857 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9969 RT: 61.03 AV:1 NL:2.27E3 T: FTMS + p NSI d Full ms2 849.90@hcd35.00 [100.00-1710.00] 200 400 600 800 1000 1200 1400 1600 m/z 0 20 40 60 80 100 Relative Abundance 1119.57568 z=1 899.47815 z=? 312.17929 z=? 411.24619 z=? 1234.60254 z=1 163.76799 z=? 785.42603 z=? 1062.55908 z=? 657.37286 z=? 1332.01221 z=? 510.31946 z=? 1513.69446 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9970 RT:61.03 AV: 1 NL: 1.23E4 T: FTMS + p NSI d Full ms2 861.94@hcd35.00 [100.00-1735.00] 200 400 600 800 1000 1200 1400 1600 m/z 0 20 40 60 80 100 Relative Abundance 1358.67700 z=1 365.19376 z=1 266.12494 z=1 860.42145 z=? 448.25320 z=1 1457.74207 z=? 1162.56665 z=1 163.76756 z=? 1015.49200 z=? 1578.91736 z=? 759.95984 z=? 589.17157 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9971 RT: 61.04 AV:1 NL:1.39E4 T: FTMS + p NSI d Full ms2 668.33@hcd35.00 [100.00-1350.00] 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 m/z 0 20 40 60 80 100 Relative Abundance 906.43085 z=1 143.11801 z=? 453.71979 z=2 1094.51331 z=1 242.15028 z=? 547.29077 z=? 876.43475 z=1 1007.48126 z=1 662.30792 z=? 400.25598 z=? 1 2 3 4 5 6 7 8 9 10 ecoli_redalk_115minrun_90mingrad_top1... 2/25/2009 9:32:13 PM ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9962 RT:61.00 AV: 1 NL: 1.99E4 T: FTMS + p NSI d Full ms2 758.42@hcd35.00 [100.00-1530.00] 200 400 600 800 1000 1200 1400 m/z 0 20 40 60 80 100 Relative Abundance 1187.64832 z=1 200.13951 z=? 594.32690 z=2 806.44598 z=1 329.18198 z=1 905.51459 z=1 1288.69727 z=1 644.84998 z=2 1090.59802 z=1 382.20880 z=? 511.25323 z=2 1467.69104 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9963 RT: 61.00 AV:1 NL:2.39E4 T: FTMS + p NSI d Full ms2 579.95@hcd35.00 [100.00-1750.00] 200 400 600 800 1000 1200 1400 1600 m/z 0 20 40 60 80 100 Relative Abundance 719.36469 z=2 589.28918 z=2 812.39856 z=2 522.61646 z=3 301.11505 z=1 866.43231 z=2 468.22293 z=2 1177.57947 z=? 1306.05542 z=? 1552.11975 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9964 RT:61.01 AV: 1 NL: 7.80E4 T: FTMS + p NSI d Full ms2 431.47@hcd35.00 [100.00-1740.00] 200 400 600 800 1000 1200 1400 1600 m/z 0 20 40 60 80 100 Relative Abundance 458.71375 z=2 581.78229 z=2 916.42065 z=1 365.19400 z=1 219.14963 z=? 815.37323 z=1 708.38354 z=1 1015.49182 z=1 1453.09192 z=? 1312.03992 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9965 RT: 61.01 AV:1 NL:1.38E4 T: FTMS + p NSI d Full ms2 727.37@hcd35.00 [100.00-1465.00] 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z 0 20 40 60 80 100 Relative Abundance 881.43732 z=1 727.36548 z=2 573.28967 z=1 487.17896 z=1 129.10245 z=? 600.26361 z=1 359.15573 z=? 967.55438 z=? 1054.58350 z=? 854.45422 z=? 1326.62781 z=1 ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9966 RT:61.02 AV: 1 NL: 1.32E4 T: FTMS + p NSI d Full ms2 558.80@hcd35.00 [100.00-1130.00] 100 200 300 400 500 600 700 800 900 1000 1100 m/z 0 20 40 60 80 100 Relative Abundance 545.30371 z=1 215.13892 z=1 874.46344 z=1 745.41852 z=1 432.22049 z=1 688.39899 z=1 816.45746 z=1 303.17639 z=? 243.13406 z=? 173.12830 z=? 915.53046 z=1 516.31775 z=1 1016.56140 z=? 615.34186 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9967 RT: 61.02 AV:1 NL:7.68E4 T: FTMS + p NSI d Full ms2 406.57@hcd35.00 [100.00-1230.00] 100 200 300 400 500 600 700 800 900 1000 1100 1200 m/z 0 20 40 60 80 100 Relative Abundance 359.20441 z=? 746.42029 z=1 647.35168 z=1 472.28802 z=1 180.10544 z=2 407.23026 z=? 718.42682 z=1 310.18143 z=2 859.50500 z=1 571.36005 z=? 931.03668 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9968 RT:61.03 AV: 1 NL: 3.96E4 T: FTMS + p NSI d Full ms2 411.91@hcd35.00 [100.00-1250.00] 100 200 300 400 500 600 700 800 900 1000 1100 1200 m/z 0 20 40 60 80 100 Relative Abundance 413.26700 z=? 545.34497 z=1 360.19949 z=1 658.42993 z=1 289.16245 z=? 217.08179 z=? 874.50885 z=? 759.47412 z=? 967.67554 z=? 1145.65857 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9969 RT: 61.03 AV:1 NL:2.27E3 T: FTMS + p NSI d Full ms2 849.90@hcd35.00 [100.00-1710.00] 200 400 600 800 1000 1200 1400 1600 m/z 0 20 40 60 80 100 Relative Abundance 1119.57568 z=1 899.47815 z=? 312.17929 z=? 411.24619 z=? 1234.60254 z=1 163.76799 z=? 785.42603 z=? 1062.55908 z=? 657.37286 z=? 1332.01221 z=? 510.31946 z=? 1513.69446 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9970 RT:61.03 AV: 1 NL: 1.23E4 T: FTMS + p NSI d Full ms2 861.94@hcd35.00 [100.00-1735.00] 200 400 600 800 1000 1200 1400 1600 m/z 0 20 40 60 80 100 Relative Abundance 1358.67700 z=1 365.19376 z=1 266.12494 z=1 860.42145 z=? 448.25320 z=1 1457.74207 z=? 1162.56665 z=1 163.76756 z=? 1015.49200 z=? 1578.91736 z=? 759.95984 z=? 589.17157 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9971 RT: 61.04 AV:1 NL:1.39E4 T: FTMS + p NSI d Full ms2 668.33@hcd35.00 [100.00-1350.00] 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 m/z 0 20 40 60 80 100 Relative Abundance 906.43085 z=1 143.11801 z=? 453.71979 z=2 1094.51331 z=1 242.15028 z=? 547.29077 z=? 876.43475 z=1 1007.48126 z=1 662.30792 z=? 400.25598 z=? ecoli_redalk_115minrun_90mingrad_top1... 2/25/2009 9:32:13 PM ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9962 RT:61.00 AV: 1 NL: 1.99E4 T: FTMS + p NSI d Full ms2 758.42@hcd35.00 [100.00-1530.00] 200 400 600 800 1000 1200 1400 m/z 0 20 40 60 80 100 Relative Abundance 1187.64832 z=1 200.13951 z=? 594.32690 z=2 806.44598 z=1 329.18198 z=1 905.51459 z=1 1288.69727 z=1 644.84998 z=2 1090.59802 z=1 382.20880 z=? 511.25323 z=2 1467.69104 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9963 RT: 61.00 AV:1 NL:2.39E4 T: FTMS + p NSI d Full ms2 579.95@hcd35.00 [100.00-1750.00] 200 400 600 800 1000 1200 1400 1600 m/z ecoli_redalk_115minrun_90mingrad_top1... 2/25/2009 9:32:13 PM ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9962 RT:61.00 AV: 1 NL: 1.99E4 T: FTMS + p NSI d Full ms2 758.42@hcd35.00 [100.00-1530.00] 200 400 600 800 1000 1200 1400 m/z 0 20 40 60 80 100 Relative Abundance 1187.64832 z=1 200.13951 z=? 594.32690 z=2 806.44598 z=1 329.18198 z=1 905.51459 z=1 1288.69727 z=1 644.84998 z=2 1090.59802 z=1 382.20880 z=? 511.25323 z=2 1467.69104 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9963 RT: 61.00 AV:1 NL:2.39E4 T: FTMS + p NSI d Full ms2 579.95@hcd35.00 [100.00-1750.00] 200 400 600 800 1000 1200 1400 1600 m/z 0 20 40 60 80 100 Relative Abundance 719.36469 z=2 589.28918 z=2 812.39856 z=2 522.61646 z=3 301.11505 z=1 866.43231 z=2 468.22293 z=2 1177.57947 z=? 1306.05542 z=? 1552.11975 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9964 RT:61.01 AV: 1 NL: 7.80E4 T: FTMS + p NSI d Full ms2 431.47@hcd35.00 [100.00-1740.00] 200 400 600 800 1000 1200 1400 1600 m/z 0 20 40 60 80 100 Relative Abundance 458.71375 z=2 581.78229 z=2 916.42065 z=1 365.19400 z=1 219.14963 z=? 815.37323 z=1 708.38354 z=1 1015.49182 z=1 1453.09192 z=? 1312.03992 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9965 RT: 61.01 AV:1 NL:1.38E4 T: FTMS + p NSI d Full ms2 727.37@hcd35.00 [100.00-1465.00] 0 20 40 60 80 100 Relative Abundance 719.36469 z=2 589.28918 z=2 812.39856 z=2 522.61646 z=3 301.11505 z=1 866.43231 z=2 468.22293 z=2 1177.57947 z=? 1306.05542 z=? 1552.11975 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9964 RT:61.01 AV: 1 NL: 7.80E4 T: FTMS + p NSI d Full ms2 431.47@hcd35.00 [100.00-1740.00] 200 400 600 800 1000 1200 1400 1600 m/z 0 20 40 60 80 100 Relative Abundance 458.71375 z=2 581.78229 z=2 916.42065 z=1 365.19400 z=1 219.14963 z=? 815.37323 z=1 708.38354 z=1 1015.49182 z=1 1453.09192 z=? 1312.03992 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9965 RT: 61.01 AV:1 NL:1.38E4 T: FTMS + p NSI d Full ms2 727.37@hcd35.00 [100.00-1465.00] 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z 0 20 40 60 80 100 Relative Abundance 881.43732 z=1 727.36548 z=2 573.28967 z=1 487.17896 z=1 129.10245 z=? 600.26361 z=1 359.15573 z=? 967.55438 z=? 1054.58350 z=? 854.45422 z=? 1326.62781 z=1 ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9966 RT:61.02 AV: 1 NL: 1.32E4 T: FTMS + p NSI d Full ms2 558.80@hcd35.00 [100.00-1130.00] 100 200 300 400 500 600 700 800 900 1000 1100 m/z 0 20 40 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z 0 20 40 60 80 100 Relative Abundance 881.43732 z=1 727.36548 z=2 573.28967 z=1 487.17896 z=1 129.10245 z=? 600.26361 z=1 359.15573 z=? 967.55438 z=? 1054.58350 z=? 854.45422 z=? 1326.62781 z=1 ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9966 RT:61.02 AV: 1 NL: 1.32E4 T: FTMS + p NSI d Full ms2 558.80@hcd35.00 [100.00-1130.00] 100 200 300 400 500 600 700 800 900 1000 1100 m/z 0 20 40 60 80 100 Relative Abundance 545.30371 z=1 215.13892 z=1 874.46344 z=1 745.41852 z=1 432.22049 z=1 688.39899 z=1 816.45746 z=1 303.17639 z=? 243.13406 z=? 173.12830 z=? 915.53046 z=1 516.31775 z=1 1016.56140 z=? 615.34186 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9967 RT: 61.02 AV:1 NL:7.68E4 T: FTMS + p NSI d Full ms2 406.57@hcd35.00 [100.00-1230.00] 100 200 300 400 500 600 700 800 900 1000 1100 1200 m/z 0 20 40 60 80 100 Relative Abundance 359.20441 z=? 746.42029 z=1 647.35168 z=1 472.28802 z=1 180.10544 z=2 407.23026 z=? 718.42682 z=1 310.18143 z=2 859.50500 z=1 571.36005 z=? 931.03668 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9968 RT:61.03 AV: 1 NL: 3.96E4 T: FTMS + p NSI d Full ms2 411.91@hcd35.00 [100.00-1250.00] 60 80 100 Relative Abundance 545.30371 z=1 215.13892 z=1 874.46344 z=1 745.41852 z=1 432.22049 z=1 688.39899 z=1 816.45746 z=1 303.17639 z=? 243.13406 z=? 173.12830 z=? 915.53046 z=1 516.31775 z=1 1016.56140 z=? 615.34186 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9967 RT: 61.02 AV:1 NL:7.68E4 T: FTMS + p NSI d Full ms2 406.57@hcd35.00 [100.00-1230.00] 100 200 300 400 500 600 700 800 900 1000 1100 1200 m/z 0 20 40 60 80 100 Relative Abundance 359.20441 z=? 746.42029 z=1 647.35168 z=1 472.28802 z=1 180.10544 z=2 407.23026 z=? 718.42682 z=1 310.18143 z=2 859.50500 z=1 571.36005 z=? 931.03668 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9968 RT:61.03 AV: 1 NL: 3.96E4 T: FTMS + p NSI d Full ms2 411.91@hcd35.00 [100.00-1250.00] 100 200 300 400 500 600 700 800 900 1000 1100 1200 m/z 0 20 40 60 80 100 Relative Abundance 413.26700 z=? 545.34497 z=1 360.19949 z=1 658.42993 z=1 289.16245 z=? 217.08179 z=? 874.50885 z=? 759.47412 z=? 967.67554 z=? 1145.65857 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9969 RT: 61.03 AV:1 NL:2.27E3 T: FTMS + p NSI d Full ms2 849.90@hcd35.00 [100.00-1710.00] 200 400 600 800 1000 1200 1400 1600 m/z 0 20 40 60 80 100 Relative Abundance 1119.57568 100 200 300 400 500 600 700 800 900 1000 1100 1200 m/z 0 20 40 60 80 100 Relative Abundance 413.26700 z=? 545.34497 z=1 360.19949 z=1 658.42993 z=1 289.16245 z=? 217.08179 z=? 874.50885 z=? 759.47412 z=? 967.67554 z=? 1145.65857 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9969 RT: 61.03 AV:1 NL:2.27E3 T: FTMS + p NSI d Full ms2 849.90@hcd35.00 [100.00-1710.00] 200 400 600 800 1000 1200 1400 1600 m/z 0 20 40 60 80 100 Relative Abundance 1119.57568 z=1 899.47815 z=? 312.17929 z=? 411.24619 z=? 1234.60254 z=1 163.76799 z=? 785.42603 z=? 1062.55908 z=? 657.37286 z=? 1332.01221 z=? 510.31946 z=? 1513.69446 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9970 RT:61.03 AV: 1 NL: 1.23E4 T: FTMS + p NSI d Full ms2 861.94@hcd35.00 [100.00-1735.00] 200 400 600 800 1000 1200 1400 1600 m/z 0 20 40 60 80 100 Relative Abundance 1358.67700 z=1 365.19376 z=1 266.12494 z=1 860.42145 z=? 448.25320 z=1 1457.74207 z=? 1162.56665 z=1 163.76756 z=? 1015.49200 z=? 1578.91736 z=? 759.95984 z=? 589.17157 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9971 RT: 61.04 AV:1 NL:1.39E4 T: FTMS + p NSI d Full ms2 668.33@hcd35.00 [100.00-1350.00] z=1 899.47815 z=? 312.17929 z=? 411.24619 z=? 1234.60254 z=1 163.76799 z=? 785.42603 z=? 1062.55908 z=? 657.37286 z=? 1332.01221 z=? 510.31946 z=? 1513.69446 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9970 RT:61.03 AV: 1 NL: 1.23E4 T: FTMS + p NSI d Full ms2 861.94@hcd35.00 [100.00-1735.00] 200 400 600 800 1000 1200 1400 1600 m/z 0 20 40 60 80 100 Relative Abundance 1358.67700 z=1 365.19376 z=1 266.12494 z=1 860.42145 z=? 448.25320 z=1 1457.74207 z=? 1162.56665 z=1 163.76756 z=? 1015.49200 z=? 1578.91736 z=? 759.95984 z=? 589.17157 z=? ecoli_redalk_115minrun_90mingrad_top10_HCD_100ng_II #9971 RT: 61.04 AV:1 NL:1.39E4 T: FTMS + p NSI d Full ms2 668.33@hcd35.00 [100.00-1350.00] 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 m/z 0 20 40 60 80 100 Relative Abundance 906.43085 z=1 143.11801 z=? 453.71979 z=2 1094.51331 z=1 242.15028 z=? 547.29077 z=? 876.43475 z=1 1007.48126 z=1 662.30792 z=? 400.25598 z=? 1.58s Base Peak Chromatogram 1x OT full scan (R=100k) + 8x parallel ddITMS2 FTMS, 1e6, R=100,000 1 2 3 4 5 6 7 8 ecoli_redalk_115minrun_90mingrad_top2... 3/22/2009 11:15:42 AM RT: 0.00000 - 119.98852 0 10 20 30 40 50 60 70 80 90 100 110 Time (min) 0 20 40 60 80 100 Relative Abundance 23491 102.65589 352.33987 23494 102.75537 352.33987 21912 88.40519 652.39581 23481 102.33362 352.33990 23497 102.85026 352.33978 21119 86.05621 594.32092 23445 101.27459 352.33963 10620 50.84714 601.96875 7249 39.38719 688.82196 11715 54.61313 514.29944 3986 27.25050 587.31500 21059 85.87886 748.44641 23505 103.01502 352.33978 14588 64.14716 581.31702 2830 22.80401 652.83569 23438 101.06048 352.34000 636 14.13973 377.21613 23750 111.10162 429.08902 NL: 1.55E7 Base Peak MS ecoli_redalk_115minr un_90mingrad_top20 _CID_SF1_100ng_II_ 100k RT: 67.29639 - 67.33411 67.300 67.305 67.310 67.315 67.320 67.325 67.330 Time (min) 0 5 10 15 20 25 30 Relative Abundance 15530 67.30279 842.41577 15533 67.31631 643.10107 15531 67.31146 358.36810 15532 67.31370 1207.68555 15536 67.32412 400.42453 15534 67.31886 790.55511 15535 67.32150 638.38196 15538 67.32915 963.67200 15528 67.29668 502.89407 15539 67.33189 775.99420 15529 67.29960 1389.90320 NL: 2.46E5 Base Peak MS ecoli_redalk_115minr un_90mingrad_top20 _CID_SF1_100ng_II_ 100k ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15530 RT: 67.30 AV: 1 NL: 2.46E5 T: FTMS + p NSI Full ms [350.00-1800.00] 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z 0 20 40 60 80 100 Relative Abundance 842.41577 R=92601 z=2 791.88892 R=96001 z=2 536.29193 R=116101 z=3 593.82440 R=109301 z=2 429.08856 R=129701 z=1 724.85583 R=99500 z=4 1200.55200 R=78600 z=2 871.41907 R=86400 z=3 967.47815 R=84400 z=2 1035.90845 R=60804 z=? 1263.40918 R=68604 z=? 1403.11755 R=65304 z=? 1126.90845 R=69004 z=? 1.58s Base Peak Chromatogram 1x OT full scan (R=100k) + 8x parallel ddITMS2 FTMS, 1e6, R=100,000 1 2 3 4 5 6 7 8 ecoli_redalk_115minrun_90mingrad_top2... 3/22/2009 11:15:42 AM RT: 0.00000 - 119.98852 0 10 20 30 40 50 60 70 80 90 100 110 Time (min) 0 20 40 60 80 100 Relative Abundance ecoli_redalk_115minrun_90mingrad_top2... 3/22/2009 11:15:42 AM RT: 0.00000 - 119.98852 0 10 20 30 40 50 60 70 80 90 100 110 Time (min) 0 20 40 60 80 100 Relative Abundance 23491 102.65589 352.33987 23494 102.75537 352.33987 21912 88.40519 652.39581 23481 102.33362 352.33990 23497 102.85026 352.33978 21119 86.05621 594.32092 23445 101.27459 352.33963 10620 50.84714 601.96875 7249 39.38719 688.82196 11715 54.61313 514.29944 3986 27.25050 587.31500 21059 23491 102.65589 352.33987 23494 102.75537 352.33987 21912 88.40519 652.39581 23481 102.33362 352.33990 23497 102.85026 352.33978 21119 86.05621 594.32092 23445 101.27459 352.33963 10620 50.84714 601.96875 7249 39.38719 688.82196 11715 54.61313 514.29944 3986 27.25050 587.31500 21059 85.87886 748.44641 23505 103.01502 352.33978 14588 64.14716 581.31702 2830 22.80401 652.83569 23438 101.06048 352.34000 636 14.13973 377.21613 23750 111.10162 429.08902 NL: 1.55E7 Base Peak MS ecoli_redalk_115minr un_90mingrad_top20 _CID_SF1_100ng_II_ 100k RT: 67.29639 - 67.33411 67.300 67.305 67.310 67.315 67.320 67.325 67.330 Time (min) 0 5 10 15 20 25 30 Relative Abundance 15530 67.30279 842.41577 15533 67.31631 643.10107 15531 67.31146 358.36810 15532 67.31370 1207.68555 15536 67.32412 400.42453 15534 67.31886 790.55511 15535 67.32150 638.38196 15538 67.32915 963.67200 15528 67.29668 502.89407 15539 67.33189 775.99420 15529 67.29960 1389.90320 NL: 2.46E5 Base Peak MS ecoli_redalk_115minr un_90mingrad_top20 _CID_SF1_100ng_II_ 100k ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15530 RT: 67.30 AV: 1 NL: 2.46E5 T: FTMS + p NSI Full ms [350.00-1800.00] 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z 0 20 40 60 80 100 Relative Abundance 842.41577 R=92601 z=2 791.88892 R=96001 z=2 536.29193 R=116101 z=3 593.82440 R=109301 z=2 429.08856 R=129701 z=1 724.85583 R=99500 z=4 1200.55200 R=78600 z=2 871.41907 R=86400 z=3 967.47815 R=84400 z=2 1035.90845 R=60804 z=? 1263.40918 R=68604 z=? 1403.11755 R=65304 z=? 1126.90845 R=69004 z=? FIGURE 6. Identification of 8 peptides from 8 consecutive ITMS2 CID spectra ran in parallel with a FT full scan at 100,000 resolution. ecoli_redalk_115minrun_90mingrad_top2... 3/22/2009 11:15:42 AM ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15531 RT: 67.31 AV: 1 T: ITMS + c NSI d Full ms2 536.29@cid30.00 [135.00-1620.00] 200 400 600 800 1000 1200 1400 1600 m/z 0 20 40 60 80 100 Relative Abundance 358.36810 519.11859 281.11957 892.62939 674.55719 982.69202 1208.66638 1376.76941 ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15532 RT: 67.31 AV: 1 T: ITMS + c NSI d Full ms2 788.89@cid30.00 [205.00-1590.00] 400 600 800 1000 1200 1400 m/z 0 20 40 60 80 100 Relative Abundance 1207.68555 644.41321 1019.63696 904.61157 370.25311 1306.74805 1402.87524 ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15533 RT: 67.32 AV: 1 T: ITMS + c NSI d Full ms2 495.60@cid30.00 [125.00-1500.00] 400 600 800 1000 1200 1400 m/z 0 50 100 Relative Abundance 643.10107 385.88620 687.47565 542.37726 970.60748 1083.57495 ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15534 RT: 67.32 AV: 1 T: ITMS + c NSI d Full ms2 632.36@cid30.00 [160.00-1275.00] 400 600 800 1000 1200 1400 m/z 0 50 100 Relative Abundance 790.55511 1017.71808 509.38208 677.41235 903.63715 559.12659 446.27826 1135.78284 ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15535 RT: 67.32 AV: 1 T: ITMS + c NSI d Full ms2 626.31@cid30.00 [160.00-1265.00] 400 600 800 1000 1200 1400 m/z 0 50 100 Relative Abundance 638.38196 417.27698 835.57587 1037.69336 892.53210 1077.70959 1250.69458 ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15536 RT: 67.32 AV: 1 T: ITMS + c NSI d Full ms2 481.78@cid30.00 [120.00-975.00] 400 600 800 1000 1200 1400 m/z 0 50 100 Relative Abundance 400.42453 589.64441 702.48511 799.60358 473.03836 864.85852 ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15537 RT: 67.33 AV: 1 T: ITMS + c NSI d Full ms2 585.84@cid30.00 [150.00-1185.00] 400 600 800 1000 1200 1400 m/z 0 50 100 Relative Abundance 500.84949 887.65942 1000.70801 644.45471 773.57709 460.26599 1071.74219 ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15538 RT: 67.33 AV: 1 T: ITMS + c NSI d Full ms2 742.90@cid30.00 [190.00-1500.00] 400 600 800 1000 1200 1400 m/z 0 50 100 Relative Abundance 963.67200 800.60016 550.94934 687.42786 1100.72498 515.34088 916.61761 1310.85999 457.26495 1 2 3 4 5 6 7 8 ecoli_redalk_115minrun_90mingrad_top2... 3/22/2009 11:15:42 AM ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15531 RT: 67.31 AV: 1 T: ITMS + c NSI d Full ms2 536.29@cid30.00 [135.00-1620.00] 200 400 600 800 1000 1200 1400 1600 m/z 0 20 40 60 80 100 Relative Abundance 358.36810 519.11859 281.11957 892.62939 674.55719 982.69202 1208.66638 1376.76941 ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15532 RT: 67.31 AV: 1 T: ITMS + c NSI d Full ms2 788.89@cid30.00 [205.00-1590.00] 400 600 800 1000 1200 1400 m/z 0 20 40 60 80 100 Relative Abundance 1207.68555 644.41321 1019.63696 904.61157 370.25311 1306.74805 1402.87524 ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15533 RT: 67.32 AV: 1 T: ITMS + c NSI d Full ms2 495.60@cid30.00 [125.00-1500.00] 400 600 800 1000 1200 1400 m/z 0 50 100 Relative Abundance 643.10107 385.88620 687.47565 542.37726 970.60748 1083.57495 ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15534 RT: 67.32 AV: 1 T: ITMS + c NSI d Full ms2 632.36@cid30.00 [160.00-1275.00] 400 600 800 1000 1200 1400 m/z 0 50 100 Relative Abundance 790.55511 1017.71808 509.38208 677.41235 903.63715 559.12659 446.27826 1135.78284 ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15535 RT: 67.32 AV: 1 T: ITMS + c NSI d Full ms2 626.31@cid30.00 [160.00-1265.00] 400 600 800 1000 1200 1400 m/z 0 50 100 Relative Abundance 638.38196 417.27698 835.57587 1037.69336 892.53210 1077.70959 1250.69458 ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15536 RT: 67.32 AV: 1 T: ITMS + c NSI d Full ms2 481.78@cid30.00 [120.00-975.00] 400 600 800 1000 1200 1400 m/z 0 50 100 Relative Abundance 400.42453 589.64441 702.48511 799.60358 473.03836 864.85852 ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15537 RT: 67.33 AV: 1 T: ITMS + c NSI d Full ms2 585.84@cid30.00 [150.00-1185.00] 400 600 800 1000 1200 1400 m/z 0 50 100 Relative Abundance 500.84949 887.65942 1000.70801 644.45471 773.57709 460.26599 1071.74219 ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15538 RT: 67.33 AV: 1 T: ITMS + c NSI d Full ms2 742.90@cid30.00 [190.00-1500.00] 400 600 800 1000 1200 1400 m/z 0 50 100 Relative Abundance 963.67200 800.60016 550.94934 687.42786 1100.72498 515.34088 916.61761 1310.85999 457.26495 1 2 3 4 5 6 7 8 ecoli_redalk_115minrun_90mingrad_top2... 3/22/2009 11:15:42 AM ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15531 RT: 67.31 AV: 1 T: ITMS + c NSI d Full ms2 536.29@cid30.00 [135.00-1620.00] 200 400 600 800 1000 1200 1400 1600 m/z 0 20 40 60 80 100 Relative Abundance 358.36810 519.11859 281.11957 892.62939 674.55719 982.69202 1208.66638 1376.76941 ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15532 RT: 67.31 AV: 1 T: ITMS + c NSI d Full ms2 788.89@cid30.00 [205.00-1590.00] 400 600 800 1000 1200 1400 m/z 0 20 40 60 80 100 Relative Abundance 400 600 800 1000 1200 1400 m/z 0 20 40 60 80 100 Relative Abundance 1207.68555 644.41321 1019.63696 904.61157 370.25311 1306.74805 1402.87524 ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15533 RT: 67.32 AV: 1 T: ITMS + c NSI d Full ms2 495.60@cid30.00 [125.00-1500.00] 400 600 800 1000 1200 1400 m/z 0 50 100 Relative Abundance 643.10107 385.88620 687.47565 542.37726 970.60748 1083.57495 ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15534 RT: 67.32 AV: 1 T: ITMS + c NSI d Full ms2 632.36@cid30.00 [160.00-1275.00] 400 600 800 1000 1200 1400 m/z 0 50 100 Relative Abundance 790.55511 1017.71808 509.38208 677.41235 903.63715 559.12659 446.27826 1135.78284 ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15535 RT: 67.32 AV: 1 T: ITMS + c NSI d Full ms2 626.31@cid30.00 [160.00-1265.00] 400 600 800 1000 1200 1400 m/z 0 50 100 Relative Abundance 638.38196 417.27698 835.57587 1037.69336 892.53210 1077.70959 1250.69458 ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15536 RT: 67.32 AV: 1 T: ITMS + c NSI d Full ms2 481.78@cid30.00 [120.00-975.00] 400 600 800 1000 1200 1400 m/z 0 50 100 Relative Abundance 400.42453 589.64441 702.48511 799.60358 473.03836 864.85852 ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15537 RT: 67.33 AV: 1 T: ITMS + c NSI d Full ms2 585.84@cid30.00 [150.00-1185.00] 400 600 800 1000 1200 1400 m/z 0 50 100 Relative Abundance 400 600 800 1000 1200 1400 m/z 0 50 100 Relative Abundance 500.84949 887.65942 1000.70801 644.45471 773.57709 460.26599 1071.74219 ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_100k #15538 RT: 67.33 AV: 1 T: ITMS + c NSI d Full ms2 742.90@cid30.00 [190.00-1500.00] 400 600 800 1000 1200 1400 m/z 0 50 100 Relative Abundance 963.67200 800.60016 550.94934 687.42786 1100.72498 515.34088 916.61761 1310.85999 457.26495 1 2 3 4 5 6 7 8 FIGURE 7. Proteins and peptides identified at <0.57% FDR (100 ng E. Coli digest, 5-40% ACN in 90 min, TOP20 CID method) 828 835 690 0 100 200 300 400 500 600 700 800 900 100ng_I 100ng_II 100 ng LTQ OT Velos LTQ OT Velos Latest QTOF technology 828 835 690 0 100 200 300 400 500 600 700 800 900 100ng_I 100ng_II 100 ng LTQ OT Velos LTQ OT Velos Latest QTOF technology 7784 7852 4917 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 100ng_I 100ng_II 100 ng LTQ OT Velos LTQ OT Velos Latest QTOF technology 7784 7852 4917 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 100ng_I 100ng_II 100 ng LTQ OT Velos LTQ OT Velos Latest QTOF technology With the new hybrid linear ion trap-orbitrap mass spectrometer, we were able to identify 72.25% more proteins and 83.83% more peptides (75-minute run, at <1% FDR) or 64.44% more proteins and 74.32% more peptides (150-minute run, at <1% FDR) than in the corresponding runs with the LTQ Orbitrap XL TM . 75’ & 150’ values: mean of 2 FIGURE 10. Proteins and peptides identified at <0.57% FDR (100 ng E. Coli digest, 5-40% ACN in 90 min, TOP10 HCD method) FIGURE 1. Schematic of the newly developed LTQ Orbitrap Velos hybrid linear ion trap-Orbitrap mass spectrometer HCD Collision Cell with Axial Field Dual Pressure Linear Ion Trap Stacked Ring Ion Guide (S‐Lens) FIGURE 11. Unique peptides (left) and proteins (right) identified at <1% FDR in two consecutive runs (1 μg C. Elegans digest, 0-40% ACN in 75 min) To further evaluate the performance of the new linear ion trap-Orbitrap mass spectrometer for characterization of complex proteomes, nanoLC-MS/MS analysis of 100 ng E.Coli digest was performed. Two different instrument methods were employed for this analysis: (1) TOP20 ITMS2 method (1 Orbitrap full scan at R=100,000 +20 data dependent CID scans), and (2) TOP10 HCD method (1 Orbitrap full scan at R=30,000 +10 data dependent HCD scans). While the first method is using the ion trap to detect the CID fragment ions, the second method employs the Orbitrap to detect the HCD fragment ions providing accurate masses for both, MS and MS/MS scans. However, using both instrument methods, the LTQ Orbitrap Velos outperformed the latest Q-TOF technology (see Fig. 7 and 10). Total of 4058 unique peptide IDs at <1% FDR 2376 831 851 1 st run 2 nd run 1 st run 2 nd run Total of 1230 protein IDs 867 180 183 1296 1921 454 LTQ OT XL LTQ OT VELOS FIGURE 12. Unique peptides identified at <1% FDR using LTQ Orbitrap XL (left) and LTQ Orbitrap Velos (right) 1 μg C. elegans digest, 0‐40% ACN in 75 min 0‐40% ACN in 75min 100ng E. coli digest 100ng E. coli digest 5‐40% ACN in 90min 5‐40% ACN in 90min 0 1000 2000 3000 4000 5000 6000 7000 5987 6221 4917 LTQ OT Velos, PT I Top10 HCD #1 Latest QTOF technology LTQ OT Velos, PT I Top10 HCD #2 0 1000 2000 3000 4000 5000 6000 7000 5987 6221 4917 0 1000 2000 3000 4000 5000 6000 7000 5987 6221 4917 LTQ OT Velos, PT I Top10 HCD #1 Latest QTOF technology LTQ OT Velos, PT I Top10 HCD #2 795 789 690 620 640 660 680 700 720 740 760 780 800 820 LTQ OT Velos, PT I Top10 HCD #1 Latest QTOF technology LTQ OT Velos, PT I Top10 HCD #2 795 789 690 620 640 660 680 700 720 740 760 780 800 820 LTQ OT Velos, PT I Top10 HCD #1 Latest QTOF technology LTQ OT Velos, PT I Top10 HCD #2 Protein IDs at <0.57% Peptide IDs at <0.57% Protein IDs at <0.57% Peptide IDs at <0.57% LTQ OT XL LTQ OT Velos 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 83.83% more unique peptide IDs