Name: synthesis and mass spectrometry analysis of oligo-peptoids
Uploaded: 2018-02-21T13:00:00.000+00:00
Duration: 11 min 44 s
Description: Watch this Scientific Journal Video about Synthesis and Mass Spectrometry Analysis of Oligo-peptoids at JoVE.com

המחברים רוצה להודות מר מייקל קונלי, ד ר רונלד צוקרמן (יציקה מולקולרית, המעבדה הלאומית לורנס ברקלי) על טכניקה תמיכה סינתזה peptoid. אנו להכיר התמיכה של הקרן הלאומית למדע (צ'ה-1301505). ספקטרומטר מסה כל הניסויים נערכו במתקן ספקטרומטר מסה של הכימיה באוניברסיטה של האוקיינוס השקט.

<ol>
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המחברים אין לחשוף.

Peptoid nonamer, Ac-Nae-(Npe-Nme)4-NH2, יש להיות מסונתז באמצעות פרוטוקול שהוצג. מנגנון סינתזה כרוך כלי דמוי מזרק פוליפרופילן מוצק-שלב התגובה מטרף מכני. כלי התגובה הם זמינים מסחרית בעלות נמוכה. מכני מטרף הוא מנגנון משותף במעבדות הכימיה. עם השימוש של כלי דמוי מזרק התגובה, פתרונות יכולים להיות נשא?...

Peptoids הם סוג של פולימרים שבשליטת רצף עם מבני עמוד השדרה מחקה את המבנה של פפטידים. Peptoids יכול להיות מסונתז מן אמינים מגוונים, המאפשרת peptoids שהפגינו מאפיינים מאוד tunable1,2. Peptoids שימשו כמודלים מולקולרי לצורך מחקר biophysical, נחשב סוכני טיפולית, תוכנן ליגנדים חלבונים3,4,5,6. Peptoids פותחו במגוון רחב של חומרים פעילים ביולוגית, כגון חומרים מימטי-נוגדן anti-עכירות, סוכני מיקרוביאלית האנזימים מעכבי7,8,9. עם אופי מאוד מסודרת, tunable, peptoids גם חשב כסוג של אחסון מידע מולקולרית10. הגילוי של יישומים מגוונים אלה דורש פיתוח שיטות אנליטיות יעיל כדי לאפיין את הרצף והמבנה של peptoids. טכניקות מבוססות ספקטרומטר מסה tandem הראו הבטחה כי השיטה של בחירה עבור ניתוח המאפיינים רצף של פולימרים שבשליטת רצף, כולל12,11,peptoids13, 14,15. עם זאת, מחקרים שיטתיים מתאם הדפוסים יון peptoid הפיצול הנובע מסה ספקטרומטר מחקרים, המידע המבני של peptoids מוגבלים מאוד.
Peptoids יכול להיות בקלות מסונתז באמצעות שיטה מעבדתי. השיטה מפותח כרוך איטראציה של שני שלבים מונומר תוספת מחזור16,17. במחזור כל תוספת, אמין מאוגד-שרף הוא acetylated על ידי haloacetic חומצה (בדרך כלל bromoacetic, BMA), זה מלווה תגובת הדחק עם אמין ראשי. למרות פרוטוקולים אוטומטיים סינתזה הוחלו באופן שגרתי של סינתזה peptoid, peptoids יכול להיות מסונתז באופן ידני עם תשואות מצוינות כימיה סטנדרטי מעבדה16,18,19, 20.
המעבדה שלנו אימצו את השיטה של סינתזה peptoid ידנית, פשוטה את המנגנון בשימוש השיטות הקיימות. בעבר למדנו את דפוסי פיצול של סדרת peptoids באמצעות MS/MS טכניקות21,22,23. התוצאות שלנו מראים כי peptoids לייצר fragmentations האופייני כאשר הם נחשפים התנגשות-induced דיסוציאציה (CID)21,23 או לכידת אלקטרון דיסוציאציה (ECD)22 ניסויים. במאמר זה נדגים כיצד oligo-peptoids יכול להיות מסונתז במעבדה לכימיה סטנדרטי, כיצד לבצע את הניסויים CID באמצעות ספקטרומטר מסה טריפל-פאול וכיצד לנתח את הנתונים ספקטרלי. Peptoid כדי להיות מסונתז והוא מאופיין הוא nonamer עם acetylation N-מסוף, amidation C-מסוף, Ac-Nae-(Npe-Nme)4-NH2. המבנה של peptoid מוצג באיור1.

<table><tbody><tr><td>ESI-triple quadrupole mass spectrometer, Varian 320L</td><td>Agilent Technologies Inc.</td><td></td><td>The mass spectrometer was acquired from Varian, Inc.</td></tr><tr><td>Varian MS workstation, Version 6.9.2, a data acquisition and data review software</td><td>Varian Inc.</td><td></td><td>The software is a part of the Varian 320L package</td></tr><tr><td>Burrell Scientific Wrist-action shaker, Model 75 DD</td><td>Fisher Scientific International Inc.</td><td>14-400-126</td><td></td></tr><tr><td>Hermle Centrifuge, Model Z 206 A</td><td>Hermle Labortechnik GmbH</td><td></td><td></td></tr><tr><td>Solid phase reaction vessel, 10 mL</td><td>Torviq</td><td>SF-1000</td><td></td></tr><tr><td>Pressure caps for reaction vessels</td><td>Torviq</td><td>PC-SF</td><td></td></tr><tr><td>Syringe filters, pore size 0.2 &amp;mu;m</td><td>Fisher Scientific Inc.</td><td>03-391-3B</td><td></td></tr><tr><td>Syringe filters, pore size 0.45 &amp;mu;m</td><td>Fisher Scientific Inc.</td><td>03-391-3A</td><td></td></tr><tr><td>Polypropylene centrifuge tuges, 50 mL</td><td>VWR International, LLC.</td><td>490001-626</td><td></td></tr><tr><td>Polypropylene centrifuge tuges, 15 mL</td><td>VWR International, LLC.</td><td>490001-620</td><td></td></tr><tr><td>ChemBioDraw, Ultra, Version 12.0</td><td>CambridgeSoft Corporation</td><td></td><td>CambridgeSoft is now part of PerkinElmer Inc.</td></tr><tr><td>Styrofoam cup, 12 Oz</td><td>Common Supermarket</td><td></td><td></td></tr><tr><td>Rink amide resin</td><td>Chem-Impex International, Inc.</td><td>10619</td><td></td></tr><tr><td>Piperidine</td><td>Chem-Impex International, Inc.</td><td>02351</td><td>Highly toxic</td></tr><tr><td>N, N&amp;rsquo;-diisopropylcarbodiimide</td><td>Chem-Impex International, Inc.</td><td>00110</td><td>Highly toxic</td></tr><tr><td>Bromoacetic acid</td><td>Chem-Impex International, Inc.</td><td>26843</td><td>Highly toxic</td></tr><tr><td>2-Phenylethylamine</td><td>VWR International, LLC.</td><td>EM8.07334.0250</td><td></td></tr><tr><td>2-Methyoxyethylamine</td><td>Sigma-Aldrich Co. LLC.</td><td>241067</td><td></td></tr><tr><td>N-Boc-ethylenediamine</td><td>VWR International, LLC.</td><td>AAAL19947-06</td><td></td></tr><tr><td>Acetic anhydride</td><td>Sigma-Aldrich Co. LLC.</td><td>252845</td><td></td></tr><tr><td>N, N-dimethylformamide</td><td>VWR International, LLC.</td><td>BDH1117-4LG</td><td>Further distillation before use</td></tr><tr><td>N, N-diisopropylethylamine</td><td>Chem-Impex International, Inc.</td><td>00141</td><td></td></tr><tr><td>Triisopropylsilane</td><td>Chem-Impex International, Inc.</td><td>01966</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Chem-Impex International, Inc.</td><td>00289</td><td>Highly toxic</td></tr><tr><td>Millipore MILLI-Q Academic Water Purification System</td><td>Millipore Corporation</td><td>ZMQP60001</td><td>For generating HPLC grade water</td></tr><tr><td>HPLC-grade Water</td><td></td><td></td><td>Produced from Millipore MILLI-Q&amp;reg; Academic Water Purification System</td></tr><tr><td>Methanol</td><td>Pharmco-Aaper</td><td>339USP/NF</td><td>HPLC grade</td></tr><tr><td>Acetonitrile</td><td>Fisher Scientific International, Inc.</td><td>A998-4</td><td>HPLC grade</td></tr><tr><td>Diethyl ether</td><td>VWR International, LLC.</td><td>BDH1121-19L</td><td>Further distillation before use</td></tr><tr><td>Dichloromethane</td><td>VWR International, LLC.</td><td>BDH1113-19L</td><td>Further distillation before use</td></tr><tr><td>Nitrogen gas</td><td>Fresno Oxygen/Barnes Supply</td><td>NIT 50-C-F</td><td>Ultra high purity, 99.9995%</td></tr><tr><td>Argon gas</td><td>Fresno Oxygen/Barnes Supply</td><td>ARG 50-C-F</td><td>Ultra high purity, 99.9995%</td></tr></tbody></table>

synthesis and mass spectrometry analysis of oligo-peptoids

Peptoids הם שבשליטת רצף מחקה-פפטיד oligomers המורכב מיחידות N alkylated גליצין. בין יישומים פוטנציאליים רבים, peptoids חשב כסוג של אחסון מידע מולקולרית. ניתוח ספקטרומטר מסה נחשב לשיטת הבחירה עבור רצף peptoids. Peptoids יכול להיות מסונתז באמצעות הכימיה מעבדתי באמצעות מעגל התגובה החוזרת בשני שלבים. כאן אנו מציגים שיטה לסנתז באופן ידני oligo-peptoids, כדי לנתח את רצף peptoids באמצעות טכניקות ספקטרומטר מסה (MS/MS) טנדם. Peptoid לדוגמה הוא nonamer המורכבת לסירוגין N-(2-methyloxyethyl) גליצין (Nme) ו N-(2-phenylethyl) גליצין (Npe), וכן של N-(2-aminoethyl) גליצין (נאה)-קצה אמיני. הנוסחה רצף של peptoid אצמנ Ac-Nae-(Npe-Nme)4-NH2, Ac קבוצת אצטיל. הסינתזה מתרחש כלי זמין מסחרית מוצק-שלב התגובה. השרף אמיד רינק משמש את התמיכה מוצק להניב את peptoid עם קבוצת אמיד-קצה קרבוקסילי. Peptoid ובמוצר נענשים רצף ניתוח באמצעות ספקטרומטר מסה טריפל-פאול מצמידים אל מקור יינון ספקטרומטריית electrospray. המדידה MS/MS מייצרת קשת של יונים שבר הנובע הדיסוציאציה של peptoid טעון. היונים פרגמנט מסודרים בהתבסס על הערכים של היחס בנפח גדול לתשלום (מ/z). הערכים מ/z של היונים פרגמנט מושווים כנגד ההמונים הנומינלי של יונים פרגמנט תיאורטית החזוי, על פי התכנית של פיצול peptoid. הניתוח יוצר תבנית פיצול של peptoid טעון. תבנית פיצול הוא מתואם רצף מונומר של peptoid נייטרלית. בהקשר זה, ניתוח MS קורא את המידע רצף של peptoids.

מבנה peptoid 9-מר עם acetylation N-מסוף, Ac-Nae-(Npe-Nme)4-NH2, מוצג באיור1. Peptoid היה מסונתז באופן ידני מגרגרי התגובה פוליפרופילן fritted דרך הגישה מעבדתי. שרף אמיד רינק (0.047 mmol, 84 מ ג עם טעינת 0.56 mmol/g) משמש את התמיכה מוצק להניב את peptoid עם amidated קצה קרבוקסילי. שרשרת peptoid זה נב?...

Watch this Scientific Journal Video about Synthesis and Mass Spectrometry Analysis of Oligo-peptoids at JoVE.com

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

סינתזה וניתוח ספקטרומטר מסה של Oligo-peptoids

פרוטוקול המתואר לסינתזה ידנית של oligo-peptoids ואחריו רצף ניתוח באמצעות ספקטרומטר מסה.

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

synthesis-and-mass-spectrometry-analysis-of-oligo-peptoids

Research

JoVE Journal

Chemistry

10.6K Views.  University of the Pacific. פרוטוקול המתואר לסינתזה ידנית של oligo-peptoids ואחריו רצף ניתוח באמצעות ספקטרומטר מסה.

Video: Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

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 ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

רצף של הצמח קיר Heteroxylans שימוש enzymic, כימית (מתילציה) הפיזיקליים (ספקטרומטריית מסה, תהודה מגנטית גרעינית) טכניקות

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

איתור מקומות לגילוי החלבון על ידי העשרה מפפטיד וספקטרומטר מסה

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Biochemistry

Synthesis and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus highly important to avoid undesired disulfide bond formation during peptide synthesis, because it may result in a completely different peptide structure, and consequently altered bioactivity. However, the correct formation of multiple disulfide bonds in a peptide is difficult to obtain by using standard self-folding methods such as conventional buffer oxidation protocols, because several disulfide connectivities can be formed. This protocol represents an advanced strategy required for the targeted synthesis of multiple disulfide-bridged peptides which cannot be synthesized via buffer oxidation in high quality and quantity. The study demonstrates the application of a distinct protecting group strategy for the synthesis of all possible 3-disulfide-bonded peptide isomers of &#181;-conotoxin PIIIA in a targeted way. The peptides are prepared by Fmoc-based solid phase peptide synthesis using a protecting group strategy for defined disulfide bond formation. The respective pairs of cysteines are protected with trityl (Trt), acetamidomethyl (Acm), and tert-butyl (tBu) protecting groups to make sure that during every oxidation step only the required cysteines are deprotected and linked. In addition to the targeted synthesis, a combination of several analytical methods is used to clarify the correct folding and generation of the desired peptide structures. The comparison of the different 3-disulfide-bonded isomers indicates the importance of accurate determination and knowledge of the disulfide connectivity for the calculation of the three-dimensional structure and for interpretation of the biological activity of the peptide isomers. The analytical characterization includes the exact disulfide bond elucidation via tandem mass spectrometry (MS/MS) analysis which is performed with partially reduced and alkylated derivatives of the intact peptide isomer produced by an adapted protocol. Furthermore, the peptide structures are determined using 2D nuclear magnetic resonance (NMR) experiments and the knowledge obtained from MS/MS analysis.

Cysteine-rich peptides fold into distinct three-dimensional structures depending on their disulfide connectivity. Targeted synthesis of individual disulfide isomers is required when buffer oxidation does not lead to the desired disulfide connectivity. The protocol deals with the selective synthesis of 3-disulfide-bonded peptides and their structural analysis using NMR and MS/MS studies.

סינתזה וקביעת מבנה ממוצע-Conotoxin Isomers PIIIA עם Connectivities דיסולפידי שונים

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus ...

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan structure are fairly conserved across all bacteria, there is also considerable variation between Gram-positives/negatives and between species. In addition, there are numerous known variations, modifications, or adaptations to the peptidoglycan that can occur within a bacterial species in response to growth phase and/or environmental stimuli. These variations produce a highly dynamic structure that is known to participate in many cellular functions, including growth/division, antibiotic resistance, and host defense avoidance. To understand the variation within peptidoglycan, the overall structure must be broken down into its constitutive parts (known as muropeptides) and assessed for overall cellular composition. Peptidoglycomics uses advanced mass spectrometry combined with high-powered bioinformatic data analysis to examine peptidoglycan composition in fine detail. The following protocol describes the purification of peptidoglycan from bacterial cultures, the acquisition of muropeptide intensity data through a liquid chromatograph&#8212;mass spectrometer, and the differential analysis of peptidoglycan composition using bioinformatics.

This protocol covers a detailed analysis of peptidoglycan composition using liquid chromatography mass spectrometry coupled with advanced feature extraction and bioinformatic analysis software.

ניתוח כמותי למחצה של פפטידוגליקן על ידי כרומטוגרפיה נוזלית, ספקטרומטריית מסה וביואינפורמטיקה

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan ...

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded oligomeric strands mediated by paired dynamic covalent interactions in a manner mimicking the thermal cycling commonly employed for the self-assembly of complementary nucleic acid sequences. Primary amine monomers bearing aldehyde and amine pendant moieties are functionalized with orthogonal protecting groups for use as dynamic covalent reactant pairs. Using a modified automated peptide synthesizer, the primary amine monomers are encoded into oligo(peptoid) strands through solid-phase submonomer synthesis. Upon purification by high-performance liquid chromatography (HPLC) and characterization by electrospray ionization mass spectrometry (ESI-MS), sequence-specific oligomers are subjected to high-loading of a Lewis acidic rare-earth metal triflate which both deprotects the aldehyde moieties and affects the reactant pair equilibrium such that strands completely dissociate. Subsequently, a fraction of the Lewis acid is extracted, enabling annealing of complementary sequence-specific strands to form information-encoded molecular ladders characterized by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The simple procedure outlined in this report circumvents kinetic traps commonly experienced in the field of dynamic covalent assembly and serves as a platform for the future design of robust, complex architectures.

A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents.

סינתזה של מידע-מיסב פטואידים והרכבה עצמית בימוי דינמי

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded ...

Sample Preparation and Relative Quantitation using Reductive Methylation of Amines for Peptidomics Studies

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the peptide biomarkers of disease or environmental stress, identifying neuropeptides, hormones, and bioactive intracellular peptides, discovering antimicrobial and nutraceutical peptides from protein hydrolysates, and can be used in studies to understand the proteolytic processes. The recent advance in sample preparation, separation methods, mass spectrometry techniques, and computational tools related to protein sequencing has contributed to the increase of the identified peptides number and peptidomes characterized. Peptidomic studies frequently analyze peptides that are naturally generated in cells. Here, a sample preparation protocol based on heat-inactivation is described, which eliminates protease activity, and extraction with mild conditions, so there is no peptide bonds cleavage. In addition, the relative quantitation of peptides using stable isotope labeling by reductive methylation of amines is also shown. This labeling method has some advantages as the reagents are commercially available, inexpensive compared to others, chemically stable, and allows the analysis of up to five samples in a single LC-MS run.

This article describes a sample preparation method based on heat-inactivation to preserve endogenous peptides avoiding degradation post-mortem, followed by relative quantitation using isotopic labeling plus LC-MS.

הכנה לדוגמה וכמות יחסית באמצעות מתילציה מופחתת של אמינים למחקרי פפטידומית

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the ...

Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take place, are inherently difficult to assess with standard biophysical methods. The introduction of mass-sensitive particle tracking (MSPT) constitutes an important step toward a thorough quantitative characterization of such processes. Technically, this was made possible through the advent of interferometric scattering microscopy (iSCAT)-based mass photometry (MP). When the background removal strategy is optimized to reveal the two-dimensional motion of membrane-associated particles, this technique allows the real-time analysis of both diffusion and molecular mass of unlabeled macromolecules on biological membranes. Here, a detailed protocol to perform and analyze mass-sensitive particle tracking of membrane-associated systems is described. Measurements performed on a commercial mass photometer achieve time resolution in the millisecond regime and, depending on the MP system, a mass detection limit down to 50 kDa. To showcase the potential of MSPT for the in-depth analysis of membrane-catalyzed macromolecule dynamics in general, results obtained for exemplary protein systems such as the native membrane interactor annexin V are presented.

This protocol describes an iSCAT-based image processing and single-particle tracking approach that enables the simultaneous investigation of the molecular mass and the diffusive behavior of macromolecules interacting with lipid membranes. Step-by-step instructions for sample preparation, mass-to-contrast conversion, movie acquisition, and post-processing are provided alongside directions to prevent potential pitfalls.

מעקב אחר חלקיקים רגישים למסה לאפיון דינמיקת מקרומולקולות הקשורות לממברנה

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take ...

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

אוליגו פרופיל המוניים (OLIMP) של סוכרים תאית

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

Biology

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.