在實驗過程中探討接合反應速率時，以每隔5 分鐘取得 MRM 面積的方法呈現酵素的
反應量值，可進一步設計以螢光共振能量轉移（Fluorescence resonance energy transfer，
FRET）的方式，藉由觀察 FRET 的螢光量值了解 Sortase A 即時的接合速率反應量值。如 以 Rhodamine-QALPETG 與 GGGGGFITC 為 受 質 ， 當 Sortase A 作 用 後 形 成 Rhodamine-QALPETGGGGGK-FITC，以 450nm 的螢光激發 FITC，若有產物則發散波長會 由530nm 轉移至 585nm，在螢光光譜上，可藉由觀察 585nm 波長的變化測得 Sortase A 接 合反應的速率 (54)。
圖 5-1：Sortase A 的 FRET 研究示意圖 (54)
而 本 研 究 試 以 Rhodamine-QALPETG 為 受 質 研 究 其 接 合 反 應 時 ， 發 現 Rhodamine-QALPETG 在 Sortase A 活性溶液（Tris5mM、NaCl 15mM、鈣離子 2 mM）中螢 光會消失，將溶液中的Tris 降低至 5μM 則螢光才會又出現，但 Sortase A 活性在此低濃度 鹽 類 環 境 中 活 性 降 低 ， 不 利 於 進 行 Sortase A 反 應 速 率 的 探 討 。 而 在 低 濃 度 鹽 類 Rhodamine-QALPETG 螢光消失的現象，未來可能是研究探討的方向。
實驗結論中顯示縮短接合反應的時間較能有效縮短 SortaseA 的作用時間，故在 β7/β8
（D185～K196）的螺旋上進行有利於辦認 GGGGG 受質的胺基酸突變，尚有很多的研究空 間。而以SrtPro 取代 SrtW 可將 GGGGGK-FITC 的胜肽段接合在修飾 LPETG 的晶片載體上，
但最佳反應條件，如 pH 值、溫度、反應時間等，仍需再做研究。且雖然 SrtPro 具有接合
的專一性，但要修飾不同的抗體在 QCM 晶片上做為探針進行免疫檢查時，將胜肽段置換
成蛋白質抗體，是否會有立體障礙，而阻礙SrtPro 的反應，仍需再做研究。
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附錄一：DNA 序列與胺基酸序列對照表
V K E Q A S K D N K Q Q A K P Q I P K D
gagaaaaccggtgtgtgggaaacccgtaaaatcttcgtggccaccgaagtgaaataagaa E K T G V W E T R K I F V A T E V K - E
SortasePro 的 DNA 與胺基酸序列
agaaggagatataccatgggcagcagccatcatcatcatcatcacagcagcggcctggtg R R R Y T M G S S H H H H H H S S G L V ccgcgcggcagccatatgcaccaccaccaccaccaccaagccaaacctcaaatcccgaaa P R G S H M H H H H H H Q A K P Q I P K gacaaaagcaaagtggcgggctatatcgaaattcctgacgccgacattaaagaacctgtg D K S K V A G Y I E I P D A D I K E P V tatccgggtccggctacttctgagcaactgaatcgtggagtgagcttcgctgaagaaaac Y P G P A T S E Q L N R G V S F A E E N gaaagcctggatgatcaaaacatcagcattgccggccacacctttatcgatcgtccgaac E S L D D Q N I S I A G H T F I D R P N tatcagttcaccaatctgaaagcggccaaaaaaggttcgatggtgtatttcaaagtcggg Y Q F T N L K A A K K G S M V Y F K V G aacgaaacccgtaaatataaaatgacctccatccgtaacgtgaaaccaacggccgtggaa N E T R K Y K M T S I R N V K P T A V E gtcctggatgaacagaaaggcaaagacaaacagctgaccctgatcacttgtgacgactat V L D E Q K G K D K Q L T L I T C D D Y aacgagaaaaccggtgtgtgggaaacccgtaaaatcttcgtggccaccgaagtgaaataa N E K T G V W E T R K I F V A T E V K -
附錄二：MALDI-TOF 分析的分子量圖譜
SrtW 的分子量為：22970.9
D165A 的分子量：22942.0
K196T 的分子量：22902.4
Srt Pro 的分子量：16643.7
附錄三：Abz-LPETG-Dnp 被 Sortase A 水 解的螢光量值對時間紀錄表
s SrtPro D165A SrtW K196T blank 0 961.3 876 887.4 561.6 165.6 1 871 724.6 694.1 485.2 347.4 2 935.2 1029 716.1 660.8 265.4 3 951.7 1151 779.6 719.3 229.2 4 1143 1129 778.7 638.9 284.1 5 1200 1186 811.1 725.2 322.6 6 1228 1179 899.9 758.3 310.5 7 1327 1257 846.9 818.2 220.3 8 1312 1539 931.8 798.8 336.9 9 1614 1574 793.2 827 268.1 10 1557 1565 980.3 879 311 11 1591 1559 969.9 747.8 332.2 12 1639 1551 963 840.2 272.5 13 1649 1591 999.4 905.3 285.8 14 1771 1685 1026 895.9 262 15 1657 1842 1022 823.3 331.7 16 1667 1795 980 941.8 297.2 17 1769 1757 1066 904.7 314.1 18 1776 1918 1120 977.1 291.4 19 1699 1994 1186 919.4 319.5 20 1779 1880 1263 893.5 415.8
21 1813 1846 1222 1046 379.9 22 1903 1705 1203 1047 344.3 23 1949 1925 1106 1044 399.7 24 1809 1751 1204 922.8 277.9 25 1604 1743 1089 1037 316 26 1753 1914 1195 1164 208.1 27 1948 1998 1177 1049 193.1 28 1739 2012 1216 984 284.5 29 1790 1821 1235 1094 302.6 30 1853 1658 1262 1110 257.6 31 1797 1850 1349 1213 305.7 32 2084 1674 1303 1168 283.5 33 2072 1994 1331 1106 278.5 34 1915 1654 1331 1135 318.4 35 1915 1837 1325 1131 284.4 36 1917 1750 1360 1137 237.6 37 2202 1926 1445 1077 267.1 38 2113 1830 1313 1080 273.1 39 2240 1843 1380 1066 222.1 40 2151 2057 1385 1145 312.9 41 2090 2096 1325 1202 308.3 42 1958 1915 1342 1144 294.7 43 2039 2035 1448 1130 358 44 2131 1849 1421 1181 362.5 45 2073 1787 1452 1190 300.2
46 1971 1811 1505 1103 322.7 47 2093 1983 1495 1048 338.8 48 1925 2062 1356 1151 393.5 49 2075 1927 1503 1219 236.3 50 1963 1883 1458 1319 317.3 51 2066 2039 1452 1143 290.6 52 2155 2169 1604 1240 369.4 53 2294 2141 1472 1195 317.2 54 1910 1913 1481 1285 327.8 55 1976 1892 1535 1235 271.2 56 2017 2319 1493 1153 328.5 57 1923 2081 1483 1261 394 58 2019 2177 1622 1275 328 59 1795 1942 1532 1202 266.7 60 1968 2205 1488 1355 361 61 2026 2294 1512 1092 246.6 62 1917 2364 1502 1178 365.8 63 1820 2276 1572 1277 265.1 64 1953 2294 1588 1254 288.9 65 1950 1812 1580 1296 353.5 66 2005 2334 1713 1376 342.2 67 2176 2147 1595 1310 331.9 68 1952 2160 1749 1202 250.7 69 1947 2062 1607 1259 269.6 70 2011 2121 1634 1158 402.3
71 2088 2144 1409 1235 239.6 72 2227 2180 1618 1310 242.6 73 2008 2192 1584 1198 279 74 1959 2272 1722 1288 342.3 75 2066 2260 1697 1266 260.2 76 2140 2244 1605 1369 299.8 77 2133 2378 1696 1336 350.8 78 1957 2451 1588 1263 330.7 79 1818 2126 1491 1373 299.5 80 2120 2373 1682 1297 287.3 81 2222 2313 1537 1393 275 82 1942 2323 1514 1338 290.8 83 1956 2152 1732 1449 262 84 2041 2095 1819 1520 312.2 85 2220 2060 1684 1324 262.4 86 1899 2163 1644 1379 290.2 87 2134 2249 1670 1305 248.1 88 1907 2329 1550 1219 278.5 89 2016 2156 1713 1322 276.5 90 1980 2241 1741 1258 335.8 91 1995 2182 1841 1299 333.4 92 2207 2239 1663 1390 430.1 93 2096 2312 1767 1286 323.4 94 2183 2126 1604 1279 362.6 95 2151 2246 1700 1389 293.3
96 2265 1981 1751 1328 337.2 97 2080 2015 1626 1407 316.5 98 1922 2171 1749 1355 269.1 99 1920 2307 1727 1289 218.8 100 1854 2421 1903 1334 276.7
附錄四：不同時間點的 MRM 的面積圖譜
SrtPro
srtPro-0
Time
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010201 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010801 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010802 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC
2013010803 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC 1.66e5 Area 10.35
24671
srtPro-20
Time
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010804 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010805 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010806 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010807 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC 3.15e5 Area 10.39
48597
SrtW
srtW-0
Time
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2012121102 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010822 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010823 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010824 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC 1.33e5 Area 10.38
19839
srtW-20
Time
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010825 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010826 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010827 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010828 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC 2.58e5 Area 10.38
40010
D165A
S165M-0
Time
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2012121107 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010217 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010218 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010219 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC 1.45e5 Area 10.52
20792
srt165-20
Time
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010811 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010221 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010813 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010814 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC 2.73e5 Area 10.39
41400
K196T
S196M-0
Time
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2012121112 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010324 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010325 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010326 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC 1.05e5 Area 10.50
15316
srt196-20
Time
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010327 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010328 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010820 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
%
0 100
2013010821 Sm (Mn, 2x3) MRM of 1 Channel ES+
TIC 2.15e5 Area 10.39
33243