ON-LINE OR OFF-LINE 2D-SDS-PAGE PROTEIN IDENTIFICATION? A COMPARISON BETWEEN CHIP/MS/MS, NANOLC/MS/MS AND MALDI-MS/MS FOLLOWING AN OPTIMISED SAMPLE PREPARATION REGIME K. Olaf Boernsen 1 , Nelson Guerreiro 1 , Georges Imbert 1 , Mark Allen 2 , Mark Baumert 2 , Reinaldo Almeida 2 1 Novartis Pharma AG, Biomarker Development, Basel, Switzerland 2 Advion BioSciences, Ltd., Norwich, UK Online or offline? Principles Figure 1: Comparison between LC and Chip - theory. If one assumes that into an HPLC system a given amount of compound will be injected. One can create a peak, for example, of 20 sec. width and with a height of 2500 counts. The resulting peak area would be 50000 sec-counts. If the same total amount of sample will be infused by the chip, one should receive the same “peak” area creating a signal over 2000 sec. long and with a height of 25 counts. 25 counts is a very weak signal, but with the chip one has approximately 10 times higher signal intensities, resulting in 250 counts peak height. This might happen because of better spray conditions on the chip. However, this is only part of the story. In proteomics directed data analysis (DDA) is used to switch automatically to MS/ MS and back again. In the picture above one has to conclude this. If 40 sec. for MS/MS would be used, this would result in an effective area of 250x40 resulting in 10000 sec. counts. With the HPLC system only 8 sec. DDA is used, resulting in 2500x8 equal to 20000 sec. counts. This is because the system needs time to measure each of the different coeluting peptides. In summary, the LC system would only be 2 times better than the chip system. Figure 2: MS/MS of 690 comparison LC & ESI Chip in detail. Sample Myoglobin digest. For the mass fragment 885 Da, we achieved 51 counts, while with the chip we achieved 12 counts. This is close to what was expected, in terms of intensity. Experimental Details Sample: Myoglobin horse digest (Michrom BioResources) Sample size: 45 fmol infused/injected Mass Spectrometer: QTOF Ultima DDA: Chip: 40 seconds (Figures 3 and 6) 60 seconds (Figure 9) LC: 8 seconds Run time: LC and Chip 35 minutes LC/MS: 75μm ID Picofrit-Column (New Objective) Infusion: ESI Chip (Advion BioSciences) Myoglobin Figure 3: MS/MS spectrum of a peptide with 690 m/z. The mass accuracy is in both cases without lock mass 16 ppm and 26 ppm. Showed no significant difference between LC and ESI Chips. Start - End Observed Mr(expt) Mr(calc) Miss Sequence 12 – 28 454.25 1812.97 1812.99 1 QISVRGLAGVENVTELK Deamidation (NQ) (No match PMF) 17 - 29 679.38 1356.75 1356.76 1 GLAGVENVTELKK (No match PMF) 34 - 41 497.79 993.57 993.58 0 HLHFTLVK (No match PMF) 278 - 289 721.85 1441.68 1441.69 0 VLYPNDNFFEGK (Ions score 49 MSMS) 352 - 358 429.26 856.50 856.50 0 ILVDLER (No match PMF) 414 - 424 559.28 1116.55 1116.56 0 VAAAFPGDVDR (No match PMF) 428 - 437 539.78 1077.54 1077.54 0 MSLVEEGAVK Oxidation (M) (Ions score 14 MSMS) 428 - 438 617.82 1233.63 1233.64 1 MSLVEEGAVKR Oxidation (M) (Ions score 0 PMF) 458 - 465 484.30 966.58 966.59 1 IHSEILKK (Ions score 9 MSMS) 521 - 532 712.87 1423.73 1423.73 0 LLSFVDDEAFIR (Ions score 37 MSMS) 555 - 568 812.44 1622.87 1622.88 0 VHINPNSLFDIQVK (Ions score 3 MSMS) 642 - 649 527.29 1052.56 1052.57 0 VIFLENYR (No match PMF) 656 - 680 822.06 2463.16 2463.18 0 VIPAADLSEQISTAGTEASGTGNMK Oxidation (M) (Ions score 33 MSMS) 800 - 810 603.80 1205.60 1205.64 1 MVIRNIATSGK Deamidation (NQ); Oxidation (M) (No match PMF) Start - End Observed Mr(expt) Mr(calc) Miss Sequence 12 – 28 454.25 1812.97 1812.99 1 QISVRGLAGVENVTELK Deamidation (NQ) (No match PMF) 17 - 29 679.38 1356.75 1356.76 1 GLAGVENVTELKK (No match PMF) 34 - 41 497.79 993.57 993.58 0 HLHFTLVK (No match PMF) 278 - 289 721.85 1441.68 1441.69 0 VLYPNDNFFEGK (Ions score 49 MSMS) 352 - 358 429.26 856.50 856.50 0 ILVDLER (No match PMF) 414 - 424 559.28 1116.55 1116.56 0 VAAAFPGDVDR (No match PMF) 428 - 437 539.78 1077.54 1077.54 0 MSLVEEGAVK Oxidation (M) (Ions score 14 MSMS) 428 - 438 617.82 1233.63 1233.64 1 MSLVEEGAVKR Oxidation (M) (Ions score 0 PMF) 458 - 465 484.30 966.58 966.59 1 IHSEILKK (Ions score 9 MSMS) 521 - 532 712.87 1423.73 1423.73 0 LLSFVDDEAFIR (Ions score 37 MSMS) 555 - 568 812.44 1622.87 1622.88 0 VHINPNSLFDIQVK (Ions score 3 MSMS) 642 - 649 527.29 1052.56 1052.57 0 VIFLENYR (No match PMF) 656 - 680 822.06 2463.16 2463.18 0 VIPAADLSEQISTAGTEASGTGNMK Oxidation (M) (Ions score 33 MSMS) 800 - 810 603.80 1205.60 1205.64 1 MVIRNIATSGK Deamidation (NQ); Oxidation (M) (No match PMF) Figure 4: The mass accuracy of the calculated peptide sequences show the same error with only on exception. This might be a wrong sequence assignment. Higher sequence coverage was observed with the ESI Chip because there were no coeluting peaks. 40 second acquisition time. ESI Chip or MALDI? Figure 5: Mass spectrum of myoglobin digest 45 fmol. Show deconvoluted ESI and MALDI spectrum. Figure 6: Comparison of MS/MS spectra between MALDI and ESI Ionization. Same peptide from 45 fmol Myoglobin digest. Table 1: Sequence coverage from different methods using the same 45 fmol (unless otherwise stated) myoglobin digest sample. It should be noted, that some doubly and triply charged peptides are measured twice. 2D-Gels Intensities below 40 mainly from dog Spot intensity = volume intensity/100 (Progenesis) Coverage vs Intensity 0 10 20 30 40 50 60 70 80 90 0 500 1000 1500 2000 2500 3000 spot intensity Coverage (%) Apolipoprotein A I Coverage vs Intensity 0 5 10 15 20 25 30 35 40 45 50 0 10 20 30 40 50 60 70 spot intensity Coverage (%) ZOOM Figure 7: Typical 2D-gel for expression profiling. In general spots which are hard to recognize against the background by the eye are the important ones. Gels are stained with colloidal coomassie blue for MS and 2 mm in diameter spots are used. Optimized ZipTip digestion method, elution with 8 mL of 30% n-Propanol, 20 ACN, 49% water with 1% acetic acid. In this way, the solution was ready to use for directly analyzing by chip-MS. Figure 9: AA sequence coverage from spots of human and dog samples. It should be noted that dog genes are limited and no additional BLAST search was performed. The spot (indicated with the blue circle) is approximately 870 times less sensitive then Apollpoprotein A1 as seen in the diagram to the left. Figure 8: Chip/MS of a typical digested 2D-gel spot; acquisition time for single MS: 10 sec. Because of the reduced ion suppression effect many peptides as doubly and triply charged ions are visible. This can also be seen in the deconvoluted spectrum of Figure 6 (Chip). • Higher sequence coverage with ESI Chip compared to LC/MS/MS • 2D-gel spot identification by ESI Chip gives good AA coverage • No time lost for fixing common nanoLC problems • 100 times less data to store • Reproducible, robust signal Conclusions Using nano-ESI there is always the question between online LC/MS/MS, direct infusion or tips filled with reverse phase material. Comparing online and offline methods the principle difference is the time for data acquisition. For LC, only a few seconds are available, but with infusion up to 60 minutes are available. This is shown in Figure 1 while Figure 2 shows experimental data. Using 8 sec DDA for LC and 40 sec DDA for infusion, equivalent coverage was acheived. In proteomics, protein identification by peptide mass fingerprinting (PMF) using MALDI is a very common, easy to use method. However, often times false positives result. MALDI or electrospray ionisation (ESI) methods with MS/MS are more accurate. Moreover, MALDI and ESI give complimentary information. With the introduction of the ESI Chip, ESI is now easy to use, reliable, robust, and provides quality data. The following work illustrates these points. Introduction LC 2 : 1 Chip time counts time counts 20 sec x 2500 counts A = 50000 sec-counts A = 50000 sec-counts 2000 sec x 25 counts 2500 counts intensity DDA 8 sec > 2500 x 8 = 20.000 sec-counts 2500 25 / 250 counts 20 sec width 2000 sec LC 2 : 1 Chip time counts time counts 20 sec x 2500 counts A = 50000 sec-counts A = 50000 sec-counts 2000 sec x 25 counts 2500 counts intensity DDA 8 sec > 2500 x 8 = 20.000 sec-counts 2500 25 / 250 counts 20 sec width 2000 sec • The NanoMate is easy to use - no instrument adjust were necessary • Reduced ion suppression when using ESI Chip • DDA could be further optimized for this application • A better combination of single MS (PMF) and MSMS data would improve data bank searches and it could increase the speed of the method. 866 868 870 872 874 876 878 880 882 884 886 888 890 892 894 896 898 900 902 904 906 908 910 912 914 916 m/z 0 100 % 0 100 % 2: TOF MSMS 689.93ES+ 51.4 885.5889 874.5251 869.5313 875.5402 886.5861 892.5438 887.5837 893.5325 894.5341 907.5560 Myoglobin_05 3 (8.489) Sm (Mn, 2x3.00); Cm (3:6) 4: TOF MSMS 689.94ES+ 12.1 885.6080 875.5114 874.5569 867.5770 886.5928 892.5502 887.6270 888.6373 893.5389 907.5491 894.5525 906.4911 903.5174 LC-MSMS Chip-MSMS Intensity Ratio: 51.4:12.1 = 4:1 Chip: 32/112 matches using 43 most intensive peaks MALDI: 15/56 matches using 54 most intensive peaks 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z 0 100 % 0 100 % 2: TOF MSMS 1378.85LD+ 25.0 1383 328 227 195 129 310 296 240 258 570 395 391 344 494 441 413 466 519 520 1382 1255 827 779 710 698 607 571 666 648 761 752 735 812 795 883 845 866 960 893 942 894 907 1238 1237 962 1120 1089 1007 1220 1365 1256 1341 1257 1318 1384 1385 1386 4: TOF MSMS 689.94ES+ 82.0 195 167 147 690 681 260 196 274 296 395 381 377 346 297 494 487 454 396 607 600 495 589 671 690 691 886 773 691 692 774 985 887 1007 1185 1084 1242 Chip: 32/112 matches using 43 most intensive peaks MALDI: 15/56 matches using 54 most intensive peaks 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z 0 100 % 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z 0 100 % 0 100 % 2: TOF MSMS 1378.85LD+ 25.0 0 100 % 2: TOF MSMS 1378.85LD+ 25.0 1383 328 227 195 129 310 296 240 258 570 395 391 344 494 441 413 466 519 520 1382 1255 827 779 710 698 607 571 666 648 761 752 735 812 795 883 845 866 960 893 942 894 907 1238 1237 962 1120 1089 1007 1220 1365 1256 1341 1257 1318 1384 1385 1386 4: TOF MSMS 689.94ES+ 82.0 1383 328 227 195 129 310 296 240 258 570 395 391 344 494 441 413 466 519 520 1382 1255 827 779 710 698 607 571 666 648 761 752 735 812 795 883 845 866 960 893 942 894 907 1238 1237 962 1120 1089 1007 1220 1365 1256 1341 1257 1318 1384 1385 1386 4: TOF MSMS 689.94ES+ 82.0 195 167 147 690 681 260 196 274 296 395 381 377 346 297 494 487 454 396 607 600 495 589 671 690 691 886 773 691 692 774 985 887 1007 1185 1084 1242 LC: 23/112 matches using 24 most intense peaks Chip: 32/112 matches using 43 most intensive peaks 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z 0 100 % 0 100 % 2: TOF MSMS 689.93ES+ 270 689.9276 195.0922 147.1172 680.9230 296.1378 260.1982 196.0977 395.2047 377.1999 494.2816 430.3098 607.3751 495.2901 690.4320 690.9366 984.6622 885.5889 772.5074 708.4189 773.5181 886.5861 892.5438 1241.7998 1184.7743 1083.7234 1119.6796 1242.8076 1243.8015 4: TOF MSMS 689.94ES+ 107 195.0947 167.0985 689.9376 680.9336 260.2089 196.1001 296.1452 395.2165 381.2303 377.2057 494.2818 487.3325 607.3719 495.2812 690.4419 690.9357 885.6080 772.5220 773.5213 984.6747 1006.5976 1184.7987 1241.8325 LC: 23/112 matches using 24 most intense peaks Chip: 32/112 matches using 43 most intensive peaks 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z 0 100 % 0 100 % 2: TOF MSMS 689.93ES+ 270 689.9276 195.0922 147.1172 680.9230 296.1378 260.1982 196.0977 395.2047 377.1999 494.2816 430.3098 607.3751 495.2901 690.4320 690.9366 984.6622 885.5889 772.5074 708.4189 773.5181 886.5861 892.5438 1241.7998 1184.7743 1083.7234 1119.6796 1242.8076 1243.8015 4: TOF MSMS 689.94ES+ 107 195.0947 167.0985 689.9376 680.9336 260.2089 196.1001 296.1452 395.2165 381.2303 377.2057 494.2818 487.3325 607.3719 495.2812 690.4419 690.9357 885.6080 772.5220 773.5213 984.6747 1006.5976 1184.7987 1241.8325 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z 0 100 % 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z 0 100 % 0 100 % 2: TOF MSMS 689.93ES+ 270 0 100 % 2: TOF MSMS 689.93ES+ 270 689.9276 195.0922 147.1172 680.9230 296.1378 260.1982 196.0977 395.2047 377.1999 494.2816 430.3098 607.3751 495.2901 690.4320 690.9366 984.6622 885.5889 772.5074 708.4189 773.5181 886.5861 892.5438 1241.7998 1184.7743 1083.7234 1119.6796 1242.8076 1243.8015 4: TOF MSMS 689.94ES+ 107 689.9276 195.0922 147.1172 680.9230 296.1378 260.1982 196.0977 395.2047 377.1999 494.2816 430.3098 607.3751 495.2901 690.4320 690.9366 984.6622 885.5889 772.5074 708.4189 773.5181 886.5861 892.5438 1241.7998 1184.7743 1083.7234 1119.6796 1242.8076 1243.8015 4: TOF MSMS 689.94ES+ 107 195.0947 167.0985 689.9376 680.9336 260.2089 196.1001 296.1452 395.2165 381.2303 377.2057 494.2818 487.3325 607.3719 495.2812 690.4419 690.9357 885.6080 772.5220 773.5213 984.6747 1006.5976 1184.7987 1241.8325 15 1 70 % MALDI 2 12 69 % MALDI 4 23 95 % LC 0 23 100 % Chip Additional matches through PMF Number of matches found by MSMS Coverage QTOF-Ultima 15 1 70 % MALDI 250 attomol 2 12 69 % MALDI 4 23 95 % LC 0 23 100 % Chip Additional matches through PMF Number of matches found by Coverage QTOF-Ultima Low abundant protein 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 m/z 0 100 % 1: TOF MS ES+ 3.61e3 x2 415.1998 523.2886 421.7587 451.2719 738.0399 737.7069 523.7932 636.0273 577.3808 636.3571 695.8612 767.3990 767.7385 789.4500 789.7943 795.3944 832.3193 957.2107 956.8821 1039.1925 957.8805 1060.1943 1060.8594 1303.6152 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 m/z 0 100 % 1: TOF MS ES+ 3.61e3 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 m/z 0 100 % 1: TOF MS ES+ 3.61e3 x2 415.1998 523.2886 421.7587 451.2719 738.0399 737.7069 523.7932 636.0273 577.3808 636.3571 695.8612 767.3990 767.7385 789.4500 789.7943 795.3944 832.3193 957.2107 956.8821 1039.1925 957.8805 1060.1943 1060.8594 1303.6152 665 670 675 680 685 690 695 700 705 710 715 720 725 m/z 0 100 % 1: TOF MS ES+ 363 695.8612 695.3657 672.8672 672.4118 668.3506 673.3652 673.8741 679.0583 678.7284 678.4838 692.3530 679.3884 681.3594 691.8373 685.3527 716.0574 696.3677 715.7185 698.0070 698.3416 700.5669 714.3425 702.3519 703.3372 707.3510 716.3854 716.7242 717.0523 723.3882 720.0305 725.3669 728.7041 665 670 675 680 685 690 695 700 705 710 715 720 725 m/z 0 100 % 1: TOF MS ES+ 363 665 670 675 680 685 690 695 700 705 710 715 720 725 m/z 0 100 % 1: TOF MS ES+ 363 695.8612 695.3657 672.8672 672.4118 668.3506 673.3652 673.8741 679.0583 678.7284 695.8612 695.3657 672.8672 672.4118 668.3506 673.3652 673.8741 679.0583 678.7284 678.4838 692.3530 679.3884 681.3594 691.8373 685.3527 716.0574 696.3677 715.7185 698.0070 698.3416 700.5669 714.3425 702.3519 703.3372 707.3510 716.3854 716.7242 717.0523 723.3882 720.0305 725.3669 728.7041 most of the peptides are visible Acknowledgements Iain Compuzano and Catherine Ryan, Micromass Guido Wahl and Stephan Carmont, Novartis 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 mass 0 100 % 1: TOF MS ES+ 5.72e4 1937 1507 1361 941 790 748 893 1087 1018 1272 1193 1379 1503 1489 1854 1607 1662 1677 1982 3404 2110 2009 3032 2501 2191 2291 2421 2681 2518 3031 2950 3369 3063 3368 3197 3405 3569 3818 3617 3936 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 mass 0 100 % 1: TOF MS ES+ 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 mass 0 100 % 1: TOF MS ES+ 5.72e4 5.72e4 1937 1507 1361 941 790 748 893 1087 1018 1272 1193 1379 1503 1489 1854 1607 1662 1677 1982 3404 2110 2009 3032 2501 2191 2291 2421 2681 2518 3031 2950 3369 3063 3368 3197 3405 3569 3818 3617 3936 MALDI Chip deconvoluted 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 mass 0 100 % 1: TOF MS LD+ 3.06e3 1361 1193 1087 941 877 1031 1272 1273 1937 1854 1362 1507 1405 1607 1662 1853 1855 1982 2110 1983 1984 2111 2501 2112 2163 2280 3962 3872 3705 2757 2585 2674 3064 3004 2822 3213 3322 3594 3355 3715 3971 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 mass 0 100 % 1: TOF MS LD+ 3.06e3 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 mass 0 100 % 1: TOF MS LD+ 3.06e3 1361 1193 1087 941 877 1031 1272 1361 1193 1087 941 877 1031 1272 1273 1937 1854 1362 1507 1405 1607 1662 1853 1855 1982 2110 1983 1984 2111 2501 2112 2163 2280 3962 3872 3705 2757 2585 2674 3064 3004 2822 3213 3322 3594 3355 3715 3971 View publication stats View publication stats