MN-S מודה תמיכה מהקרן הלאומית למדע, רוי להעניק מל&quot;ג 1011859. המחברים תודה להכיר מתקן שלישיית ספקטרומטריית מסה באוניברסיטת צפון קרוליינה בגרינסבורו לשימוש בספקטרומטר המסה תרמו פישר סיינטיפיק LTQ Orbitrap XL. המחברים מודים דניאל טוד, וינסנט סיקה, וBrandie Erhmann באוניברסיטת צפון קרוליינה בגרינסבורו להצעות מועילות והערות לגבי עבודה זו.

הערה: נא להתייעץ כל גיליונות הנתונים של בטיחות חומרים הרלוונטיים (MSDS) לפני השימוש. כלוריד מרקורי הוא חומר כימי רעיל. ציוד מגן אישי (כפפות, משקפי בטיחות, ומעבדה מעיל) חייבים להיות משוחק בעת המסירה ואת כל הפתרונות הקשורים. השלך פתרונות בבקבוקי פסולת כימיים מסומנים באופן ברור המיועדים למתכות כבדות. 1. הכנה של 5 מ&quot;מ degassed אמוניום Formate המאגר, pH 7.5 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ממיסים .1576 גרם של חיץ formate אמוניום ב450 מיליליטר של מים כיתה HPLC. התאם את ה- pH של התמיסה מעל עם 1 חומצה פורמית M ואמוניום הידרוקסיד 1 M 7.5. העבר את הפתרון הזה לבקבוק נפח 500 מיליליטר ולהוסיף מים HPLC לקו הכיול לעשות פתרון formate אמוניום 5 מ&quot;מ. </li><li style=";text-align:right;direction:rtl"> דגה חיץ formate אמוניום 5 מ&quot;מ מתחת מערכת ואקום במשך 10 דקות וטיהור עם ארגון. חזור פעמיים ופתרון חנות תחת ארגון. ביום שימוש, לסנן את פתרון החיץ דרך פילטר 0.2 מיקרון beforשימוש בדואר. </li></ol> 2. הכנה של מרקורי פתרונות כלוריד (II) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לשקול את .2375 כספית גרם (II) כלוריד. לפזר אותו ב 25 מיליליטר של חיץ formate אמוניום 5 מ&quot;מ לייצר (II) פתרון כלוריד 0.035 M כספית. </li><li style=";text-align:right;direction:rtl"> להוסיף .214 מיליליטר של 0.035 פתרון M הכספית כלוריד (II) ל9.785 חיץ formate אמוניום מיליליטר של 5 מ&quot;מ כדי ליצור פתרון M 7.5 x 10 -4. שמיכת פתרון 7.5 x 10 -4 M כספית (II) עם גז ארגון. </li></ol> 3. הכנת CGGC פתרון במלאי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ממיסים 2.0 מ&quot;ג של tetrapeptide dicysteinyl, CGGC, ב0.118 מיליליטר של אצטוניטריל הכיתה HPLC ולאחר מכן להוסיף 1.0647 מיליליטר של 5 מ&quot;מ formate אמוניום, pH 7.5 חיץ שכבר degassed בארגון להניב פתרון מניות מ&quot;מ CGGC 5. </li><li style=";text-align:right;direction:rtl"> להוסיף 225 μl של פתרון מניות מ&quot;מ CGGC 5 ל1,275 μl של pH היווצרות אמוניום 5 מ&quot;מ 7.5 חיץ לתת פתרון M CGGC 7.5 x 10 -4. </li></ol> 4. הכנהשל תערובות תגובה שונות של מרקורי (II) וCGGC <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכנה של 1: 0.5 יחס של כספית (II): פתרון CGGC <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הנח 255 μl של formate אמוניום 5 מ&quot;מ, חיץ pH 7.5 לתוך צינור microcentrifuge 1.5 מיליליטר. הוסף 30 μl של פתרון כספית כלוריד (II) 7.5 x10 -4 M לתוך צינור microcentrifuge 1.5 מיליליטר עם חיץ formate אמוניום. </li><li style=";text-align:right;direction:rtl"> מערבולת הפתרון במשך 10 שניות. ואז להוסיף 15 μl של 7.5 x 10 -4 פתרון M CGGC לmicrocentrifuge 1.5 מיליליטר. מערבולת הפתרון במשך 10 שניות. תן הפתרון לעמוד במשך 10 דקות לפני ההזרקה לתוך ספקטרומטר המסה. </li></ol></li><li style=";text-align:right;direction:rtl"> הכנה של 1: 1 יחס של כספית (II): פתרון CGGC <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הנח 240 μl של formate אמוניום 5 מ&quot;מ, חיץ pH 7.5 לתוך צינור microcentrifuge 1.5 מיליליטר. הוסף 30 μl של פתרון כספית כלוריד (II) 7.5 x10 -4 M לתוך צינור microcentrifuge 1.5 מיליליטר עם חיץ formate אמוניום. </li><li style=";text-align:right;direction:rtl"> מערבולת הפתרון במשך 10 שניות. אזלהוסיף 30 μl של 7.5 x 10 -4 פתרון M CGGC לתוך צינור microcentrifuge 1.5 מיליליטר. חזור באופן דומה כמפורט בסעיף 4.1. </li></ol></li><li style=";text-align:right;direction:rtl"> הכנת 1: 2 יחס של כספית (II): פתרון CGGC <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הנח 210 μl של formate אמוניום 5 מ&quot;מ, חיץ pH 7.5 לתוך צינור microcentrifuge 1.5 מיליליטר. הוסף 30 μl של פתרון כספית כלוריד (II) 7.5 x10 -4 M לתוך צינור microcentrifuge 1.5 מיליליטר עם חיץ formate אמוניום. </li><li style=";text-align:right;direction:rtl"> מערבולת הפתרון במשך 10 שניות. ואז להוסיף 60 μl של 7.5 x 10 -4 פתרון M CGGC לתוך צינור microcentrifuge 1.5 מיליליטר. חזור באופן דומה כמפורט בסעיף 4.1. </li></ol></li></ol> 5. הכנת CEEC פתרון במלאי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ממיסים 3.5 מ&quot;ג של tetrapeptide dicysteinyl, CEEC, ב0.145 מיליליטר של אצטוניטריל הכיתה HPLC לפזר פפטיד. לאחר מכן להוסיף 13.067 מיליליטר של 5 formate אמוניום מ&quot;מ, pH 7.5 חיץ שכבר degassed בארגון לייצר 0.5 מ &#39;פתרון M CEEC. </li><li style=";text-align:right;direction:rtl"> מערבולת הפתרון עד שכל פפטיד נמס. להוסיף 1.125 מיליליטר של 0.5 מ&quot;מ פתרון CEEC ו0.375 מיליליטר של 5 מ&quot;מ formate אמוניום, חיץ pH 7.5 לצינור 1.5 מיליליטר microcentrifuge לתת פתרון x 10 -5 M CEEC 7.5. מערבולת עד מעורב. </li></ol> 6. הכנת תערובות תגובה שונות של מרקורי (II) ופתרון CEEC <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכנה של 1: 0.5 יחס של הכספית (II): פתרון CEEC <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הנח 255 μl של formate אמוניום 5 מ&quot;מ, חיץ pH 7.5 לתוך צינור microcentrifuge 1.5 מיליליטר. הוסף 30 μl של פתרון כספית כלוריד (II) 7.5 x10 -4 M לתוך צינור microcentrifuge 1.5 מיליליטר עם חיץ formate אמוניום. </li><li style=";text-align:right;direction:rtl"> מערבולת הפתרון במשך 10 שניות. ואז להוסיף 15 μl של 7.5 x 10 -4 פתרון M CEEC לתוך צינור microcentrifuge 1.5 מיליליטר. חזור באופן דומה כמפורט בסעיף 4.1. </li></ol></li><li style=";text-align:right;direction:rtl"> הכנה של 1: 1 יחס של כספית (II): פתרון CEEC <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הנח 24081; L של formate אמוניום 5 מ&quot;מ, חיץ pH 7.5 לתוך צינור microcentrifuge 1.5 מיליליטר. הוסף 30 μl של פתרון כספית כלוריד (II) 7.5 x10 -4 M לתוך צינור microcentrifuge 1.5 מיליליטר עם חיץ formate אמוניום. </li><li style=";text-align:right;direction:rtl"> מערבולת הפתרון במשך 10 שניות. ואז להוסיף 30 μl של 7.5 x 10 -4 פתרון M CEEC לתוך צינור microcentrifuge 1.5 מיליליטר. חזור באופן דומה כמפורט בסעיף 4.1. </li></ol></li><li style=";text-align:right;direction:rtl"> הכנת 1: 2 יחס של כספית (II): פתרון CEEC <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הנח 210 μl של formate אמוניום 5 מ&quot;מ, חיץ pH 7.5 לתוך צינור microcentrifuge 1.5 מיליליטר. הוסף 30 μl של פתרון כספית כלוריד (II) 7.5 x10 -4 M לתוך צינור microcentrifuge 1.5 מיליליטר עם חיץ formate אמוניום. </li><li style=";text-align:right;direction:rtl"> מערבולת הפתרון במשך 10 שניות. ואז להוסיף 60 μl של 7.5 x 10 -4 פתרון M CEEC לתוך צינור microcentrifuge 1.5 מיליליטר. חזור באופן דומה כמפורט בסעיף 4.1. </li></ol></li></ol> 7. Analyזיע תערובות התגובה של מרקורי (II) ודוגמאות CGGC ידי Orbitrap ESI ספקטרומטריית המסה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכנת ספקטרומטר מסת ESI 16 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> צייר של סטנדרטים כיול 100 μl לתוך מזרק זכוכית 500 μl. </li><li style=";text-align:right;direction:rtl"> מניחים את המזרק בעריסת המזרק של משאבת MS, לצרף את צינורות, ולהזריק לספקטרופוטומטר ההמוני. </li><li style=";text-align:right;direction:rtl"> להגדיר את שם הקובץ לריצה על ידי בחירת סמל הקובץ ולהקליד את שם הקובץ. </li><li style=";text-align:right;direction:rtl"> בחר כפתור נתונים לרכוש במודול רכישת נתונים ולאסוף 150 סריקות. </li><li style=";text-align:right;direction:rtl"> ניתוח chromatogram כדי לאמת את הסטנדרטים כיול על ידי פתיחת מודול עיבוד נתונים של התוכנה. פתח את מודול, ללכת להגיש תפריט ובחר באפשרות &quot;לפתוח&quot;, ובחר את הקובץ בתיבת הדו-שיח. ודא שהפסגות בchromatogram כדי לתאם את המסה לחייב יחסים של הסטנדרטים. </li><li style=";text-align:right;direction:rtl"> נקה את מזרק זכוכית 500 μl על ידי ציור את מתנול הכיתה HPLC 500 μl ואז לוותר מ &#39;אתנול לתוך כוס. </li><li style=";text-align:right;direction:rtl"> צייר את 500 μl של מתנול הכיתה HPLC לתוך מזרק הזכוכית ולשטוף את המערכת לפי צעד 7.1.2. </li><li style=";text-align:right;direction:rtl"> בחר את שיטת התקנת מודול של התוכנה כדי להגדיר את הפרמטרים. בחר את תפריט מצב הסריקה ולזהות המנתח כFTMS, ולאחר מכן לחץ על &quot;אישור&quot;. לאחר מכן על ידי לחיצה על הסמלים השונים בדף ספקטרום תצוגה בזמן אמת, להגדיר את הפרמטרים הבאים: זרימת גז שיעור נדן: 10, טמפרטורת מקור: 0, מתח נימים: 37 V, עדשת Tube: 95 V, מתח ריסוס: 4.20 קילו וולט , קצב זרימה 10.00 μl / דקה, מנתח: FTMS, מספר סריקות: 150. </li></ol></li><li style=";text-align:right;direction:rtl"> ריצת דגימות CGGC על ספקטרומטר מסת ESI <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הפעל את חיץ pH formate אמוניום 5 מ&quot;מ 7.5. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הנח 500 μl של חיץ formate אמוניום 5 מ&quot;מ לתוך מזרק זכוכית 500 μl, למקם אותו לתוך עריסת המזרק של משאבת MS, ולצרף את צינורות. </li><li style=";text-align:right;direction:rtl"> הפעל חיץ דרך צינורות במשך 1-2 דקות. </li><li style=";text-align:right;direction:rtl"> להגדיר את שם הקובץ לריצהעל ידי בחירת סמל הקובץ ולהקליד את שם הקובץ. </li><li style=";text-align:right;direction:rtl"> בחר כפתור נתונים לרכוש במודול ולאסוף 150 סריקות. </li><li style=";text-align:right;direction:rtl"> לחץ על לחצן הפעלה כדי לעצור אוסף אחרי 150 סריקות נאספות. </li><li style=";text-align:right;direction:rtl"> פתח את מודול דפדפן נתונים, ואז ללכת להגיש תפריט ובחר באפשרות &quot;לפתוח&quot;, ובחר את הקובץ בתיבת הדו-שיח. ודא שאין בפסגות 483, 683, 1,163 ו1,363 מ &#39;/ z נמצאות דומה לפפטיד או כספית (II) מתחמי -peptide. </li></ol></li><li style=";text-align:right;direction:rtl"> הפעל 1: כספית 0.5 (השני): פתרון יחס CGGC. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הנח 250 μl של 1: 0.5 כספית (II): יחס CGGC של המדגם לתוך המזרק. </li><li style=";text-align:right;direction:rtl"> מניחים את המזרק לתוך תושבת המזרק של משאבת MS, לצרף את צינורות, וראש המנגנון. </li><li style=";text-align:right;direction:rtl"> בחר שם קובץ עבור הטווח על ידי בחירת סמל הקובץ ולהקליד את שם הקובץ. </li><li style=";text-align:right;direction:rtl"> לחץ על כפתור נתונים לרכוש במודול רכישת נתונים ולאסוף 150 סריקות ולחץ על לחצן הפעלה כדי לעצור את האוסף. </li><li style=";text-align:right;direction:rtl"> פתח את מודול, ללכת להגיש תפריט ובחר באפשרות &quot;לפתוח&quot;, ובחר את הקובץ בתיבת הדו-שיח. ודא שchromatogram מכיל פסגות כוללים אחד לפפטיד CGGC לבד. </li><li style=";text-align:right;direction:rtl"> שטוף את המזרק על ידי aspirating עם חיץ formate אמוניום 500 μl ולאחר מכן מחלק חיץ formate אמוניום לתוך כוס. </li><li style=";text-align:right;direction:rtl"> בחר בלחצן הפסולת בטרשת הנפוצה ולשטוף את צינורות שלוש פעמים עם 500 חיץ formate אמוניום μl. </li><li style=";text-align:right;direction:rtl"> שטוף את המזרק על ידי aspirating עם מתנול 500 μl ולאחר מכן מחלק מתנול לתוך כוס. </li><li style=";text-align:right;direction:rtl"> לשטוף פעם אחת צינורות עם 500 מתנול μl. </li><li style=";text-align:right;direction:rtl"> כפתור גלאי עומס בחר בטרשת הנפוצה. </li><li style=";text-align:right;direction:rtl"> הוסף 500 μl של חיץ formate אמוניום למזרק. </li><li style=";text-align:right;direction:rtl"> מניחים את המזרק לתוך תושבת המזרק של משאבת MS, לצרף את צינורות, וראש המנגנון. </li><li style=";text-align:right;direction:rtl"> בחר שם קובץ עבור המאגר מנוהל על ידי בחירת סמל הקובץ ולהקליד את שם הקובץ. </li><li style=";text-align:right;direction:rtl"> לִלְחוֹץכפתור נתונים לרכוש ולאסוף 150 סריקות ולאחר מכן לחץ על לחצן הפעלת התחנה. </li><li style=";text-align:right;direction:rtl"> פתח את מודול דפדפן נתונים, ללכת להגיש תפריט ובחר באפשרות &quot;לפתוח&quot;, ובחר את הקובץ בתיבת הדו-שיח. ודא שchromatogram הוא חלל של פסגות מהמ&quot;כ הקודמת: ריצת CGGC. </li></ol></li><li style=";text-align:right;direction:rtl"> הפעל 1: כספית 1 (השני): פתרון יחס CGGC. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הנח 250 μl של 1: 1 כספית (II): מדגם יחס CGGC לתוך המזרק. </li><li style=";text-align:right;direction:rtl"> חזור באופן דומה כפי שתואר לצעדים 7.2.2.2 ל7.2.2.15. </li></ol></li><li style=";text-align:right;direction:rtl"> הפעל 1: 2 הכספית (II): פתרון יחס CGGC <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הנח 250 μl של 1: 2 כספית (II): מדגם ריכוז CGGC לתוך המזרק. </li><li style=";text-align:right;direction:rtl"> חזור באופן דומה כפי שתואר לצעדים 7.2.2.2 ל7.2.2.15. </li></ol></li></ol></li></ol> 8. ניתוח תערובות התגובה של מרקורי ודוגמאות CEEC ידי Orbitrap ESI ספקטרומטריית המסה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ריצת דגימות CEEC על ספקטרומטר מסת ESI <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הליך ניתוח חוזר (צעדים 7.1-7.2) באמצעות דגימות CEEC ותערובות תגובה של כספית (II) וCEEC ביחסי stoichiometric שונים. </li></ol></li></ol>

<ol><li>Clifton, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mercury+exposure+and+public+health%5BTitle%5D)+AND+%22Pediatr.+Clin.+N.+Am%22%5BJournal%5D)">Mercury exposure and public health.</a> Pediatr. Clin. N. Am. 54, 237-269 (2007).</li>
<li>Andersen, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Principles+and+Recent+Developments+in+Chelation+Treatment+of+Metal+Intoxication%5BTitle%5D)+AND+%22Chem.+Rev%22%5BJournal%5D)">Principles and Recent Developments in Chelation Treatment of Metal Intoxication.</a> Chem. Rev. 99, 2683-2710 (1999).</li>
<li>Aposhian, H. V., Maiorino, R. M., Gonzalez-Ramirez, D., Zuniga-Charles, M., Xu, Z., Hurlbut, J. M., Junco-Munoz, P., Dart, R. C., Aposhian, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mobilization+of+heavy+metals+by+newer%2C+therapeutically+useful+chelating+agents%5BTitle%5D)+AND+%22Toxicology%22%5BJournal%5D)">Mobilization of heavy metals by newer, therapeutically useful chelating agents.</a> Toxicology. 97, 23-38 (1995).</li>
<li>Flora, S. J. S., Pachauri, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chelation+in+Metal+Intoxication%5BTitle%5D)+AND+%22Int.+J.+Environ.+Res.+Public+Health%22%5BJournal%5D)">Chelation in Metal Intoxication.</a> Int. J. Environ. Res. Public Health. 7, 2745-2788 (2010).</li>
<li>Campbell, J. R., Clarkson, T. W., Omar, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+therapeutic+use+of+2%2C3-dimercaptopropane-1-sulfonate+in+two+cases+of+inorganic+mercury+poisoning%5BTitle%5D)+AND+%22JAMA%22%5BJournal%5D)">The therapeutic use of 2,3-dimercaptopropane-1-sulfonate in two cases of inorganic mercury poisoning.</a> JAMA. 256, 3127-3130 (1986).</li>
<li>Rooney, J. P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+thiols%2C+dithiols%2C+nutritional+factors+and+interacting+ligands+in+the+toxicology+of+mercury%5BTitle%5D)+AND+%22Toxicology%22%5BJournal%5D)">The role of thiols, dithiols, nutritional factors and interacting ligands in the toxicology of mercury.</a> Toxicology. 234, 145-156 (2007).</li>
<li>Lin, X., Brooks, J., Bronson, M., Ngu-Schwemlein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evalution+of+the+association+of+mercury+%28II%29+with+some+dicysteinyl+tripeptides%5BTitle%5D)+AND+%22Bioorg.+Chem%22%5BJournal%5D)">Evalution of the association of mercury (II) with some dicysteinyl tripeptides.</a> Bioorg. Chem. 44, 8-18 (2012).</li>
<li>Ngu-Schwemlein, M., Lin, X., Rudd, B., Bronson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthesis+and+ESI+mass+spectrometric+analysis+of+the+association+of+mercury%28II%29+with+multi-cysteinyl+peptides%5BTitle%5D)+AND+%22J.+Inorg.+Biochem%22%5BJournal%5D)">Synthesis and ESI mass spectrometric analysis of the association of mercury(II) with multi-cysteinyl peptides.</a> J. Inorg. Biochem. 133, 8-23 (2014).</li>
<li>Winther, J. R., Thorpe, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+thiols+and+disulfides%5BTitle%5D)+AND+%22Biochimica+et.+Biophysica+Acta%22%5BJournal%5D)">Quantification of thiols and disulfides.</a> Biochimica et. Biophysica Acta. 1840, 838-846 (2014).</li>
<li>D'Agstino, A., Colton, R., Traeger, J. C., Cantry, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+Electrospray+Mass+Spectrometric+Study+of+Organomercury+%28II%29+and+Mercuric+Interactions+with+Peptides+Involving+Cysteinyl+Ligands%5BTitle%5D)+AND+%22Eur.+Mass+Spectrom%22%5BJournal%5D)">An Electrospray Mass Spectrometric Study of Organomercury (II) and Mercuric Interactions with Peptides Involving Cysteinyl Ligands.</a> Eur. Mass Spectrom. , 273-285 (1990).</li>
<li>Hofstadler, S. A., Sannes-Lowery, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Applications+of+ESI-MS+in+drug+discovery%3A+interrogation+of+noncovalent+complexes%5BTitle%5D)+AND+%22Nature+Reviews+Drug+Discovery%22%5BJournal%5D)">Applications of ESI-MS in drug discovery: interrogation of noncovalent complexes.</a> Nature Reviews Drug Discovery. 5, 585-595 (2006).</li>
<li>Rubino, F. M., Verduci, C., Giampiccolo, R., Pulvirenti, S., Brambilla, G., Columbi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+Characterization+of+Homo-+and+Heterodimeric+Mercury+%28II%29-bis-thiolates+of+Some+Biologically+Relevant+Thiols+by+Electrospray+Ionization+and+Triple+Quadruple+Tandem+Mass+Spectrometry%5BTitle%5D)+AND+%22J.+Am.+Soc.+Mass+Spectrom%22%5BJournal%5D)">Molecular Characterization of Homo- and Heterodimeric Mercury (II)-bis-thiolates of Some Biologically Relevant Thiols by Electrospray Ionization and Triple Quadruple Tandem Mass Spectrometry.</a> J. Am. Soc. Mass Spectrom. 15, 288-300 (2003).</li>
<li>Krupp, E. M., Milne, B. F., Mestrot, A., Meharg, A. A., Feldmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Investigation+into+mercury+bound+to+biothiols%3A+structural+identification+using+ESI-ion-trap+MS+and+introduction+of+a+method+for+their+HPLC+separation+with+simultaneous+detection+by+ICP-MS+and+and+ESI-MS%5BTitle%5D)+AND+%22Anal.+Bioanal.+Chem%22%5BJournal%5D)">Investigation into mercury bound to biothiols: structural identification using ESI-ion-trap MS and introduction of a method for their HPLC separation with simultaneous detection by ICP-MS and and ESI-MS.</a> Anal. Bioanal. Chem. 390, 1753-1764 (2008).</li>
<li>Schaumlöffel, D., Tholey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+directions+of+electrospray+mass+spectrometry+for+elemental+speciation+analysis%5BTitle%5D)+AND+%22Anal.+Bioanal.+Chem%22%5BJournal%5D)">Recent directions of electrospray mass spectrometry for elemental speciation analysis.</a> Anal. Bioanal. Chem. 400, 1645-1652 (2011).</li>
<li>Patiny, L., Borel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ChemCalc%3A+a+building+block+for+tomorrow%27s+chemical+infrastructure%5BTitle%5D)+AND+%22J.+Chem.+Inf.+Model%22%5BJournal%5D)">ChemCalc: a building block for tomorrow's chemical infrastructure.</a> J. Chem. Inf. Model. 53, 1223-1228 (2013).</li>
<li>Thermo Scientific. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Xcaibur+Versions+2.1.0-2.3.0+Data+Acquisition+and+Processing+User+Guide.+Revision+E.+United+States%22">Xcaibur Versions 2.1.0-2.3.0 Data Acquisition and Processing User Guide. Revision E. United States</a>. , Thermo Fisher Scientific Inc. (2012).</li>
<li>Falcone, G., Foti, C., Gianguzza, A., Giuffrè, O., Napoli, A., Pettignano, A., Piazzese, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sequestering+ability+of+some+chelating+agents+towards+methylmercury%28II%29%5BTitle%5D)+AND+%22Anal.+Bioanal.+Chem%22%5BJournal%5D)">Sequestering ability of some chelating agents towards methylmercury(II).</a> Anal. Bioanal. Chem. 405 (2), 881-893 (2013).</li>
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There is no disclosure for the work reported.

Tetrapeptide dicysteinyl הידרופובי CGGC (10 C H 18 N O 4 5 S 2; MW = 338) (איור 1), יוצר קומפלקסים עם כספית (II) כפי שמוצג באיור 2 ולוח 1 בנוסף, הוא יוצר הדימרים פפטיד וtrimers. בהדרגה בסכום של עליות פפטיד בתערובת התגובה. כפי שניתן לראות על ידי ער?...

Current clinical drugs prescribed for chelation therapy of mercury poisoning1 contain thiol group(s), which is/are responsible for binding and sequestering mercury ions2,3. However, studies have shown that these small thiol compounds [dimercaptosuccinic acid (DMSA) and dimercaptopropane-sulfonic acid (DMPS)] are not optimal for mercury chelation therapy4-6. Therefore, there is a need to understand the association and complex formation tendencies of mercury with thiols to enhance the rational drug design of thiol compounds for mercury chelation. Recently, we reported that n-alkyl and aryl dicysteinyl tripeptides with dithiol groups can serve as effective "double anchors" to accommodate the coordination sites of mercury(II) to form 1:1 mercury(II):peptide and 1:2 mercury(II):(peptide)2 complexes7. Additionally, we studied the effect of increasing cysteinyl residues on complex type formations8. In this study, we investigate the association of mercury(II) with two dicysteinyl tetrapeptides, where the cysteinyl residues are separated by two amino acid residues. In order to evaluate the effect of auxiliary binding groups for mercury, the intervening amino acids are either two glycine (unsubstituted) residues or two glutamic acid (gamma-carboxylated) residues. 
The reaction of cysteinyl peptide with mercury(II) requires conditions that will prevent the oxidation of the cysteinyl thiol groups to form disulfide bonds9. Moreover, the association of mercury(II) with cysteinyl peptides to form various types of mercury-peptide complexes is dependent on the initial ratio of mercury(II): peptide in the reaction mixture7,8. The types of mercury-peptide complexes formed in these reaction mixtures can be analyzed by soft-ionization mass spectroscopy, which is a sensitive analytical tool for determining species interactions between metal ions and peptides10-14. Accordingly, it will provide a profile of the various types of mercuriated peptide adducts that are formed under a specified electrospray ionization condition. Here, we will show how cysteinyl peptides and mercury(II) chloride solutions can be prepared in degassed ammonium formate buffer solution blanketed with argon to minimize oxidation. By reacting varying mole equivalents of mercury(II) with dicysteinyl tetrapeptides, we will show how the initial ratio of mercury(II):peptide has an effect on the types of complexes formed. We will also show how electrospray ionization (ESI) mass spectrometry can be used as a characterization tool to provide an accurate stoichiometry of mercury to peptide in the complexes formed. The associated video protocol will demonstrate the experimental conditions for preparing the mercury complexes, the procedure for analyzing the reaction mixtures under specified electrospray ionization conditions, and the characterization of the stoichiometries of mercury(II)-dicysteinyl tetrapeptide complexes, based on the distinct mercury isotope distribution patterns, by using the ChemCal program15. It is intended to assist those who are interested in using ESI orbitrap mass spectrometry to analyze various complexes formed by metal ions that exist in different isotopic forms.

<table><tbody><tr><td>Mercury(II) chloride</td><td>Sigma-Aldrich</td><td>429724</td><td>Highly toxic</td></tr><tr><td>Ammonium formate</td><td>Sigma-Aldrich</td><td>516961</td><td></td></tr><tr><td>Formic acid</td><td>Sigma-Aldrich</td><td>F0507</td><td></td></tr><tr><td>Ammonium hydroxide</td><td>Fisher</td><td>A512-P500</td><td></td></tr><tr><td>HPLC water</td><td>Fisher</td><td>W5-4</td><td></td></tr><tr><td>HPLC Acetonitrile</td><td>Fisher</td><td>BP2405-1</td><td></td></tr><tr><td>HPLC Methanol</td><td>Fisher</td><td>A452-4</td><td></td></tr></tbody></table>

a study of the complexation of mercury(ii) with dicysteinyl tetrapeptides by electrospray ionization mass spectrometry

In this study we evaluated a method for the characterization of complexes, formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptide, by electrospray ionization orbitrap mass spectrometry. This strategy is based on previous successful characterization of mercury-dicysteinyl complexes involving tripeptides by utilizing mass spectrometry among other techniques. Mercury(II) chloride and a dicysteinyl tetrapeptide were incubated in a degassed buffered medium at varying stoichiometric ratios. The complexes formed were subsequently analyzed on an electrospray mass spectrometer consisting of a hybrid linear ion- and orbi- trap mass analyzer. The electrospray ionization mass spectrometry (ESI-MS) spectra were acquired in the positive mode and the observed peaks were then analyzed for distinct mercury isotopic distribution patterns and associated monoisotopic peak. This work demonstrates that an accurate stoichiometry of mercury and peptide in the complexes formed under specified electrospray ionization conditions can be determined by using high resolution ESI MS based on distinct mercury isotopic distribution patterns.

<html> מחקר בוצע כדי לאפיין את ההרכב המורכב כספית-פפטיד האפשרי לשתי tetrapeptides, CGGC וCEEC (איור 1) על ידי ספקטרומטריית מסת ESI. מתחמים של כספית (II) עם CGGC או CEEC נחקרו על ידי מגיב התערובות של כספית (II) ופתרונות פפטיד בשלושה יחסים שונים טוחנות: 1: 0.5, 1: 1, ו 1: 2 (כס?...

Watch this Scientific Journal Video about A Study of the Complexation of MercuryII with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry at JoVE.com

A Study of the Complexation of MercuryII with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

The characterization of complexes formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptides by electrospray ionization orbitrap mass spectrometry is presented.

חקר complexation של מרקורי (II) עם Dicysteinyl Tetrapeptides ידי Ionization electrospray ספקטרומטריית המסה

In this study we evaluated a method for the characterization of complexes, formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptide, ...

a-study-complexation-mercury-ii-with-dicysteinyl-tetrapeptides

Research

JoVE Journal

Chemistry

A Study of the Complexation of Mercury(II) with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

7.9K Views.  University of North Carolina at Greensboro. The characterization of complexes formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptides by electrospray ionization orbitrap mass spectrometry is presented.

Video: A Study of the Complexation of MercuryII with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

מכלולי פפטיד הטרנסממברני ייצור של דיסולפיד-התייצבו ללימודים מבניים

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

Compact Quantum Dots for Single-molecule Imaging

Single-molecule imaging is an important tool for understanding the mechanisms of biomolecular function and for visualizing the spatial and temporal heterogeneity of molecular behaviors that underlie cellular biology 1-4. To image an individual molecule of interest, it is typically conjugated to a fluorescent tag (dye, protein, bead, or quantum dot) and observed with epifluorescence or total internal reflection fluorescence (TIRF) microscopy. While dyes and fluorescent proteins have been the mainstay of fluorescence imaging for decades, their fluorescence is unstable under high photon fluxes necessary to observe individual molecules, yielding only a few seconds of observation before complete loss of signal. Latex beads and dye-labeled beads provide improved signal stability but at the expense of drastically larger hydrodynamic size, which can deleteriously alter the diffusion and behavior of the molecule under study.
Quantum dots (QDs) offer a balance between these two problematic regimes. These nanoparticles are composed of semiconductor materials and can be engineered with a hydrodynamically compact size with exceptional resistance to photodegradation 5. Thus in recent years QDs have been instrumental in enabling long-term observation of complex macromolecular behavior on the single molecule level. However these particles have still been found to exhibit impaired diffusion in crowded molecular environments such as the cellular cytoplasm and the neuronal synaptic cleft, where their sizes are still too large 4,6,7.
Recently we have engineered the cores and surface coatings of QDs for minimized hydrodynamic size, while balancing offsets to colloidal stability, photostability, brightness, and nonspecific binding that have hindered the utility of compact QDs in the past 8,9. The goal of this article is to demonstrate the synthesis, modification, and characterization of these optimized nanocrystals, composed of an alloyed HgxCd1-xSe core coated with an insulating CdyZn1-yS shell, further coated with a multidentate polymer ligand modified with short polyethylene glycol (PEG) chains (Figure 1). Compared with conventional CdSe nanocrystals, HgxCd1-xSe alloys offer greater quantum yields of fluorescence, fluorescence at red and near-infrared wavelengths for enhanced signal-to-noise in cells, and excitation at non-cytotoxic visible wavelengths. Multidentate polymer coatings bind to the nanocrystal surface in a closed and flat conformation to minimize hydrodynamic size, and PEG neutralizes the surface charge to minimize nonspecific binding to cells and biomolecules. The end result is a brightly fluorescent nanocrystal with emission between 550-800 nm and a total hydrodynamic size near 12 nm. This is in the same size range as many soluble globular proteins in cells, and substantially smaller than conventional PEGylated QDs (25-35 nm).

We describe the preparation of colloidal quantum dots with minimized hydrodynamic size for single-molecule fluorescence imaging. Compared to conventional quantum dots, these nanoparticles are similar in size to globular proteins and are optimized for single-molecule brightness, stability against photodegradation, and resistance to nonspecific binding to proteins and cells.

נקודות קוונטיות קומפקטיות להדמית מולקולה בודדה

Single-molecule imaging is an important tool for  understanding the mechanisms of biomolecular function and for visualizing the  spatial and temporal ...

Engineering

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1H/2H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g. crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex. &#160;&#160;

Protein conformation and dynamics are key to understanding the relationship between protein structure and function. Hydrogen exchange coupled with high-resolution mass spectrometry is a versatile method to study the conformational dynamics of proteins as well as characterizing protein-ligand and protein-protein interactions, including contact interfaces and allosteric effects.

ניתוח דינמיקת חלבון באמצעות מימן Exchange ספקטרומטריית מסה

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

המרת מימן-דאוטריום electrospray יינון זמן נפתרה ספקטרומטריית מסה לימוד מבנה החלבון Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Biochemistry

Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for characterizing lyophilized formulations of protein therapeutics. Labeling followed by suitable proteolytic digestion allows the protein structure and interactions to be mapped with peptide-level resolution. Since the protein structural elements are stabilized by a network of chemical bonds from the main-chains and side-chains of amino acids, specific labeling of atoms in the amino acid residues provides insight into the structure and conformation of the protein. In contrast to routine methods used to study proteins in lyophilized solids (e.g., FTIR), ssHDX-MS and ssPL-MS provide quantitative and site-specific information. The extent of deuterium incorporation and kinetic parameters can be related to rapidly and slowly exchanging amide pools (Nfast, Nslow) and directly reflects the degree of protein folding and structure in lyophilized formulations. Stable photolytic labeling does not undergo back-exchange, an advantage over ssHDX-MS. Here, we provide detailed protocols for both ssHDX-MS and ssPL-MS, using myoglobin (Mb) as a model protein in lyophilized formulations containing either trehalose or sorbitol.

Here, we present detailed protocols for solid-state amide hydrogen/deuterium exchange mass spectrometry (ssHDX-MS) and solid-state photolytic labeling mass spectrometry (ssPL-MS) for proteins in solid powders. The methods provide high-resolution information on protein conformation and interactions in the amorphous solid-state, which may be useful in formulation design.

Mass spectrometric גישות לחקר מבנה חלבון ואינטראקציות באבקות Lyophilized

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for ...

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide&#8217;s mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide&#8217;s acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin.

Ion mobility-mass spectrometry and molecular modeling techniques can characterize the selective metal chelating performance of designed metal-binding peptides and the copper-binding peptide methanobactin. Developing new classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance.

שיטות לקביעת מבנה ומנגנונים של זיהוי יון מתכת וחמצון פעילות של כריכת מתכת Oligopeptides

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, ...

Analysis of Complex Molecules and Their Reactions on Surfaces by Means of Cluster-Induced Desorption/Ionization Mass Spectrometry

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass spectrometry (MS) of complex molecules and their reactions on surfaces. DINeC is based on a beam of SO2 clusters impacting on the sample surface at low cluster energy. During cluster-surface impact, some of the surface molecules are desorbed and ionized via dissolvation in the impacting cluster; as a result of this dissolvation-mediated desorption mechanism, low cluster energy is sufficient and the desorption process is extremely soft. Both surface adsorbates and molecules of which the surface is composed of can be analyzed. Clear and fragmentation-free spectra from complex molecules such as peptides and proteins are obtained. DINeC does not require any special sample preparation, in particular no matrix has to be applied. The method yields quantitative information on the composition of the samples; molecules at a surface coverage as low as 0.1 % of a monolayer can be detected. Surface reactions such as H/D exchange or thermal decomposition can be observed in real-time and the kinetics of the reactions can be deduced. Using a pulsed nozzle for cluster beam generation, DINeC can be efficiently combined with ion trap mass spectrometry. The matrix-free and soft nature of the DINeC process in combination with the MSn capabilities of the ion trap allows for very detailed and unambiguous analysis of the chemical composition of complex organic samples and organic adsorbates on surfaces.

Neutral SO2 clusters of low kinetic energy (&#60; 0.8 eV/constituent) are used to desorb complex surface molecules such as peptides or lipids for further analysis by means of mass spectrometry using an ion trap mass spectrometer. No special sample preparation is required, and real-time observation of reactions is possible.

ניתוח של מולקולות מורכבות והתגובות שלהם על משטחים באמצעות מצבר-המושרה באמצעות אשכול/אינון המסה ספקטרומטריה

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass ...

Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of protein therapeutics and the determination of the conformational transition pathways critical for biological functions, ranging from molecular recognition to enzymatic catalysis. Hydrogen/deuterium exchange (HDX) reaction coupled with top-down mass spectrometric (MS) analysis provides a means to characterize protein higher-order structures and dynamics in a conformer-specific manner. The conformational resolving power of this technique is highly dependent on the efficiencies of separating protein states at the intact protein level and minimizing the residual non-deuterated protic content during the HDX reactions. 
Here we describe a capillary electrophoresis (CE)-based variant of the HDX MS approach that aims to improve the conformational resolution. In this approach, proteins undergo HDX reactions while migrating through a deuterated background electrolyte solution (BGE) during the capillary electrophoretic separation. Different protein states or proteoforms that coexist in solution can be efficiently separated based on their differing charge-to-size ratios. The difference in electrophoretic mobility between proteins and protic solvent molecules minimizes the residual non-deuterated solvent, resulting in a nearly complete deuterating environment during the HDX process. The flow-through microvial CE-MS interface allows efficient electrospray ionization of the eluted protein species following a rapid mixing with the quenching and denaturing modifier solution at the outlet of the sprayer. The online top-down MS analysis measures the global deuteration level of the eluted intact protein species, and subsequently, the deuteration of their gas-phase fragments. This paper demonstrates this approach in differential HDX for systems, including the natural protein variants coexisting in milk.

Presented here is a protocol for a capillary electrophoresis-based hydrogen/deuterium exchange (HDX) approach coupled with top-down mass spectrometry. This approach characterizes the difference in higher-order structures between different protein species, including proteins in different states and different proteoforms, by conducting concurrent differential HDX and electrophoretic separation.

חילופי מימן/דיטריום מבוססי נימי אלקטרופורזה לאפיון קונפורמציה של חלבונים עם ספקטרומטריית מסה מלמעלה למטה

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

ניתוח מערכות חלבון גדול על ידי ספקטרומטריית מסה מבנית

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

Biology

Thermochemical Studies of Ni(II) and Zn(II) Ternary Complexes Using Ion Mobility-Mass Spectrometry

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry (ES-IM-MS) and energy-resolved threshold collision-induced dissociation (TCID) to measure the thermochemistry of the dissociation of negatively-charged [amb+M(II)+NTA]- ternary complexes into two product channels: [amb+M(II)] + NTA or [NTA+M(II)]-&#160;+ amb, where M = Zn or Ni and NTA is nitrilotriacetic acid. The complexes contain one of the alternative metal binding (amb) heptapeptides with the primary structures acetyl-His1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7 or acetyl-Asp1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7, where the amino acids&#39; Aa1,2,6,7 positions are the potential metal-binding sites. Geometry-optimized stationary states of the ternary complexes and their products were selected from quantum chemistry calculations (presently the PM6 semi-empirical Hamiltonian) by comparing their electronic energies and their collision cross-sections (CCS) to those measured by ES-IM-MS. From the PM6 frequency calculations, the molecular parameters of the ternary complex and its products model the energy-dependent intensities of the two product channels using a competitive TCID method to determine the threshold energies of the reactions that relate to the 0 K enthalpies of dissociation (&#916;H0). Statistical mechanics thermal and entropy corrections using the PM6 rotational and vibrational frequencies provide the 298 K enthalpies of dissociation (&#916;H298). These methods describe an EI-IM-MS routine that can determine thermochemistry and equilibrium constants for a range of ternary metal ion complexes.

Thermochemical Studies of NiII and ZnII Ternary Complexes Using Ion Mobility-Mass Spectrometry

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry, semi-empirical quantum calculations, and energy-resolved threshold collision-induced dissociation to measure the relative thermochemistry of the dissociation of related ternary metal complexes.