Name: preparation of homogeneous maldi samples for quantitative applications
Uploaded: 2016-10-28T13:00:00.000+00:00
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This work is supported by the Genomics Research Center, Academia Sinica and the Ministry of Science and Technology of Taiwan, the Republic of China (Contract No. 104-2119-M-001-014).

NOT: Bu protokol, kurutuldu-damlacık yöntemiyle hazırlanan maltotrioz ve bradikinin fragmanı (1-7) mekansal heterojen azaltmak için geliştirilmiştir. Protokol hazırlanması ve ön koşullandırma, örnek bırakma ve kurutma ve kütle spektrometrisi veri analizi de dahil olmak üzere, üç ana adımdan oluşur. prosedürleri tarif ve aşağıda daha ayrıntılı olarak tarif edilmiştir: 1. Hazırlık ve Önkoşullanma <ol><li> Örnek Plate Temizleme <ol><li> nitril eldiven giyin ve deterjan ve distile-deiyonize su (DDW) ile hafifçe örnek plaka el yıkayın. </li><li> metanol (MeOH) ve DDW ile örnek plaka durulayın. </li><li> 600 ml beher örnek plaka takın ve DDW ile doldurun. </li><li> ultrasonik banyoda (200 W, 40 kHz) &#39;de 15 dakika süre ile DDW içinde örnek plaka sonikasyon. </li><li> kaptan DDW çıkarın ve MeOH ile beher doldurun. </li><li> ultrasonik banyo (200 W, 40 kHz) 15 dakika boyunca MeOH içerisinde örnek plaka sonikasyon. </li><li> Azot gazı ile plaka üzerinde çözücü damla uçururum ve örnek birikimi önce kuru numune plaka tutun. </li></ol></li><li> Düzenlenmesi Kurutma Odası Sıcaklığı NOT:. Kurutma odası 35 x 20 x 45 cm 3 (W x D x Y) akrilik haznesi Şekil 1, bu kurutma sisteminin resmini gösterir. odası kurutma bölmesinin içine monte kalibre edilmiş bir higrometre ile gözlenmiş düşük nispi nem durumunu korumak için sabit bir akış oranında bir gaz akış ölçer ile oda sıcaklığında azot gazı ile temizlenmiştir. programlanmış sabit sıcaklık su sirkülatör sahip kurutma bölmesi bir bakır temel blok çelik örneği plakaları karşılamak için kullanılır. bakır taban bloğu 25 ° C 5 örnek plaka ısısını düzenlemek için yapabiliyor. hava, bakır taban bloğu ve numune plakasının sıcaklık K-tipi termokupllar ile kontrol edilir. <ol><li> Kapıyı açın ve hızla bakır örnek plaka koymakBaz bloğu sonra kapağını kapatın. </li><li> El ile saatte 10 standart kübik feet (SCFH&#39;dir) azot akış hızını ayarlamak için gaz debi ölçer ayarlayın. </li><li> bağıl nem her zaman% 25&#39;in altına sağlamak için higrometre ve ince ayar gaz debimetre ile kurutma odasında bağıl nemi izleyin. </li><li> K-tipi termokupl tarafından numune plaka sıcaklığını izlemek ve örnek plakası deney ya da kontrol için oda sıcaklığında (25 ° C) 5 ° C ulaşana kadar manuel olarak su sirkülasyon sıcaklığını ayarlamak. NOT: tasarlanan sıcaklıkta örnek plaka stabilize etmek için, su sirkülasyon sıcaklığı tipik olarak tasarlanmış örnek daha düşük, 5 ° C ila 0 olarak ayarlanır. Örneğin, örnek plakasına 5 ° C tutmak için su sirkülatörün sıcaklık ayarı 2 ° C ila 0 aralığındadır; 25 ° C&#39;de bir örnek plaka korumak için, suyun sirkülatörün sıcaklık ayarı 23-25 ​​° C aralığındadır. </li><li> Gerekli sıcaklık ve bağıl nem örnek birikimi önce (Tablo 1) ulaşılır emin olun. Not: farklı numune plakası sıcaklıkları kurutma işlemleri için tüm parametrelerin yanı sıra ayar değerleri Tablo 1 &#39;de gösterilmiştir. NOT: oda kapısı uzun süre açık ise düşük örnek plaka sıcaklığında numune plaka üzerinde su yoğunlaşması oluşabilir. Su yoğunlaşması oluşursa kapıyı kapatın ve su yoğunlaşması kurumuş kadar üzerinde herhangi bir örnek yatırmak ETMEYİN. </li></ol></li><li> Matris ve analit Çözümleri Hazırlanması <ol><li> matris çözümleri hazırlanması <ol><li> % 50 DDW sulu solüsyon: 50% asetonitril (ACN) ile 0.1 M thap çözeltisi hazırlayın. </li></ol></li><li> Analitlerin hazırlanması <ol><li> DDW ile 10 -4 M maltotriozdur çözeltisi hazırlayın. </li><li> % 50 asetonitril, 10 5 M bradikinin fragmanı hazırlandı (1-7) çözeltisi(ACN):% 50 DDW sulu çözelti. </li></ol></li></ol></li></ol> 2. Örnek Biriktirme ve Kurutma <ol><li> Karışımlar 0.1M thap solüsyonu 0.25 | il 10 4 M maltotrioz veya 10 5 M bradikinin fragmanı (1-7), bir mikrosantrifüj tüpü içinde çözeltiler 0.25 ul. </li><li> 3 sn karışık bir çözüm karıştırın. </li><li> santrifüj tüpünün dibinde çözüm toplamak için 2 sn (2000 xg) karışık bir çözüm santrifüjleyin. </li><li> dikkatle, kurutma odasının kapısını açın pipet ile örnek plaka üzerinde solüsyonun 0.1 ul yatırmak ve hemen kapıyı kapatın. </li><li> Örnek damlacık kurumasını bekleyin. . NOT: Farklı numune plaka sıcaklıkları genellikle gözlenen kurutma süreleri Tablo 1&#39;de listelenen 5 ° C örnek plaka sıcaklığı için, ortalama kuruma süresi 800 1000 sn; 25 ° C Örnek plakası sıcaklığı için ortalama kuruma süresi 100 ile 150 sec. </li><li> Kuruduktan sonra, kurutma odasının kapısını açın. </li><li> oda sıcaklığında (25 ° C) su sirkülasyon sıcaklığını ayarlamak. NOT: Numune plaka kurutma işlemi sırasında, oda sıcaklığında (25 ° C) sürekli tutulursa, bu adımı atlayın. </li><li> Oda sıcaklığında (25 ° C) Örnek plaka sıcaklığı döndükten sonra, kurutma bölmesinin örnek plakasını çıkarın. </li><li> 5X stereomikroskop altında örnek morfolojisi incelemek ve bir anlık parlak alan görüntü almak. Not: kristal morfolojileri olarak beklenmemektedir, bu aynı prosedür ile yeni bir numune hazırlamak için gereklidir. Tipik bir kristal morfolojisi, Şekil 2&#39;nin üst panel gösterilmiştir. NOT: 5 ° C gibi düşük örnek plaka sıcaklığına sahip durumlarda, kurutma bölmesinin çıkarmadan önce oda sıcaklığına örnek plaka ısıtmak için önemlidir. Örnekleri yatırma yaparken, önceden karıştırılmış Solutio tutmak YAPMAYINn 10 sn boyunca pipet ucu. numuneleri yatırma sonra yeniden premix çözüm kullanmayan ETMEYİN. Şekil 2&#39;nin üst panel farklı numune plakası sıcaklıkları ile hazırlanan numunelerin parlak alan görüntüleri gösterir. </li></ol> 3. Kütle Spektrometresi Veri Analizi <ol><li> Kütle Spektrometre Veri Toplama NOT: hazırlandıktan sonra, numune görüntüleme kütle spektrometresi kullanılarak analiz edilebilir. Bu çalışmada, görüntüleme MS deneyler laboratuar yönelik yapılmış senkronlaştırılmış çift kutup TOF kullanılarak gerçekleştirilir (DP-TOF) kütle spektrometresi görüntüleme. Görüntüleme yeteneği ile 15 ticari MALDI-TOF kütle spektrometre aynı zamanda, deneyler için de uygundur. kütle spektrometresi doğrusal ekstraksiyon ve optimize edilmiş emme gecikmeler ile pozitif iyon modunda çalıştırılır. iyonların kinetik enerjisi 20 kV. Lazer ışını boyutu örnek yüzeyinde çapı 35 mikron ve her nokta spektrum ave olduğunu5 lazer çekim öfke. <ol><li> MALDI kütle spektrometresi içine örnek plaka yerleştirin. </li><li> adımlarda 2,1-2,9 hazırlanan örnek kütle spektrometresi analizi görüntüleme gerçekleştirin. </li><li> sonuç penceresinde gösterilen kitle listeden karakteristik kitle tepe seçin ve iki boyutlu iyon görüntü çizmek için &quot;2D&quot; tıklayın. Not: thap ile karıştırılır maltotrioz için, karakteristik tepe noktaları maltotrioz, protonlanmış thap sodiated ve thap sodiated edilir. Thap ile karıştırıldı bradikinin fragmanı (1-7) için, karakteristik tepe noktaları bradikinin fragmanını (1-7) protonlanmış thap protonlanmış ve thap sodiated içerir. </li><li> sinyal yoğunluğu üst ve alt limitlerini belirlemek için pop-up pencere ayar düğmelerini tıklatın ve &quot;bir resmi kaydetmek&quot; butonuna tıklayınız. Bu ayar, iyon resimlerin kontrastını tanımlar. NOT: veri her bireyin setinde, kırık bölgeler ve düşük parlaklığını gösteren boş noktalar ortadan kalkar. </li><li> Gözlemlemek ve iyon karşılaştırmakAdım 2.9 olarak alınmıştır parlak alan görüntü ile görüntü. Not: kütle spektrometrisi ve özellikle iyonlarının görüntülerin inşaat Görüntüleme ticari araçlar ile elde edilebilir. Nedeniyle veri toplama ve analiz yazılımı çeşitli kullanıcıların yüksek kaliteli görüntüler elde etmek için cihaz satıcısı tarafından sağlanan yazılım talimatları takip etmelidir. </li></ol></li><li> Veri analizi NOT: Numunelerin heterojen kantitatif analiz edilir. Bu gösteri, her numune iyonları dağılımını analiz etmek in-house geliştirilen yazılımın birden fazla eş merkezli bölüme ayrılmıştır. analiz, tek başına bir veri analiz yazılımı kullanılarak gerçekleştirilebilir. <ol><li> önemsiz alanları kaldırmak için sonuç penceresinde gösterilen iyon görüntüde boş noktalar ve kırık bölgeleri tıklayın. NOT: Bu prosedür iyon görüntünün temel alanını tanımlar. </li><li> iyon görüntünün dıştaki katman bulmak için lütfen &quot;kenar bulmak&quot; tıklayın. </li><li> bir veritabanında dış tabakasında iyon bolluğu bilgileri kaydetmek ve aynı anda iyon görüntüden bu katmanı kaldırmak için &quot;düşeriz&quot; tıklayın. Bu dıştaki katman temsil eden bir onay kutusu sonucu penceresinin &quot;çıkış verileri&quot; listesinde görünür. </li><li> iyon görüntünün merkez tanımlanır yineleyerek 3.2.2 ve 3.2.3 adımları. </li><li> Tıklayın ve &quot;çıkış verileri&quot; listedeki tüm onay kutularını seçin ve verileri vermek için &quot;ihracat&quot; a tıklayın. </li><li> iyonların mekansal dağılımı bilgilerini elde etmek için her katmanın ortalama iyon bolluğu hesaplamak için tablo yazılımını kullanarak dışa aktarılan verileri açın. </li></ol></li></ol>

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The authors declare no competing financial interest.

önceki teorik tahminlere dayanarak, damlacıklar içindeki sıcaklık kaynaklı hidrodinamik akışları dışa çözücü buharlaştırma yoluyla uyarılan kılcal akışları üstesinden gelebilir. sıcaklık damlacık artışı içinde gradyanlar zaman moleküllerin bu iç sirkülasyonu verimliliği artırılmıştır. çevre sıcaklığında çevresi muhafaza edilirken, 5 ° C&#39;nin altında Örnek plaka sıcaklığı tutmak zaman tahmin edilen sonuçlara göre, damlacık içindeki sirkülasyon akımlarının ort...

Mass spectrometry (MS) is one of the most important analytical techniques for analyzing the molecular compositions of complex samples. Among all the ionization methods used in MS, matrix-assisted laser desorption/ionization (MALDI) is the most sensitive and widely used method in bioanalytical applications.1 In comparison to other ionization techniques, MALDI has the highest sensitivity and high tolerance to salt contaminants. Such analytical properties make MALDI the first choice for carbohydrate analysis and many proteomics applications. However, sample preparation is a crucial step for obtaining high quality data in MALDI-MS.
The most commonly used sample preparation method for MALDI-MS is the dried-droplet method, in which sample droplets are deposited on a surface and dried under ambient conditions. This drying method is simple and generally effective.2-5 However, a common problem in the dried-droplet method is that the resultant analyte/matrix crystals normally distribute irregularly. In many cases, the crystals aggregate at the periphery of sample areas, resulting in the so-called ring-stain formation.6-8 The heterogeneous crystal morphologies affect the spatial distribution of analyte molecules, which results in severe fluctuation in ion signal over sample areas. Such severe signal fluctuations and poor data reproducibility are known as the "sweet spot" problem in MALDI-MS.9 Thus, there is a great need for reducing spatial heterogeneities in MALDI-MS dried droplet applications.
Hydrodynamic flows in the sample droplet play an important role in determining the spatial distribution of samples prepared with the dried-droplet method.10-12 It was found that the evaporation of solvent induces outward capillary flows within droplets, which are responsible for the ring-stain formation.7,10 In contrast, recirculation flows induced by tangential surface-tension gradients may counterbalance the outward capillary flows.13 If the recirculation flow speeds are higher than that of the outward capillary flows, the samples can be efficiently redistributed to reduce the heterogeneity problem.14
In this work, we demonstrate a detailed protocol for preparing samples with a simple drying chamber to induce efficient recirculation flows during droplet drying processes. Droplet drying conditions are precisely controlled, including the temperatures of the sample plate and its surroundings, and the relative humidity within the chamber. The model analytes include maltotriose and bradykinin chain (1-7). The matrix used for the demonstration is 2,4,6-trihydroxyacetophenone (THAP). The samples are examined with time-of-flight (TOF) MS, and the data are analyzed quantitatively to show the reduction of heterogeneity.

<table><tbody><tr><td>&lt;strong&gt;Reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Detergent powder</td><td>Alconox</td><td>242985</td><td></td></tr><tr><td>Methanol</td><td>Merck</td><td>106009</td><td></td></tr><tr><td>Acetonitrile</td><td>Merck</td><td>100003</td><td></td></tr><tr><td>2,4,6-trihydroxyacetophenone (THAP)</td><td>Sigma-Aldrich</td><td>T64602&amp;nbsp;</td><td></td></tr><tr><td>Bradykinin fragment (1-7)</td><td>Sigma-Aldrich</td><td>B1651</td><td></td></tr><tr><td>Maltotriose</td><td>Sigma-Aldrich</td><td>47884</td><td></td></tr><tr><td>Pipette tips</td><td>Mettler Toledo</td><td>17005091</td><td></td></tr><tr><td>Microcentrifuge tube</td><td>Axygen</td><td>MCT-150-C</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Milli-Q water purification system</td><td>Millipore</td><td>ZMQS6VFT1</td><td></td></tr><tr><td>Powder-free nitrile gloves</td><td>Microflex</td><td>SU-690</td><td></td></tr><tr><td>600 ml beaker</td><td>Duran</td><td>2110648</td><td></td></tr><tr><td>Ultrasonic cleaner</td><td>Delta</td><td>DC300H</td><td></td></tr><tr><td>Hygrometer</td><td>Wisewind</td><td>5330</td><td></td></tr><tr><td>Nitrogen gas flowmeter</td><td>Dwyer</td><td>RMA-6-SSV</td><td></td></tr><tr><td>K-type thermocouples</td><td>Digitron</td><td>311-1670</td><td></td></tr><tr><td>Centrifuge</td><td>Select BioProducts</td><td>Force Mini&amp;nbsp;</td><td></td></tr><tr><td>Pipette</td><td>Rainin</td><td>pipet-lite XLS</td><td></td></tr><tr><td>Stereomicroscope</td><td>Olympus</td><td>SZX16</td><td></td></tr><tr><td>Temperature controllable drying chamber</td><td>this lab</td><td></td><td></td></tr><tr><td>Synchronized dual-polarity time-of-flight imaging mass spectrometer (DP-TOF IMS)</td><td>this lab</td><td></td><td></td></tr><tr><td>MALDI-TOF stainless steel sample target</td><td>this lab</td><td></td><td></td></tr></tbody></table>

preparation of homogeneous maldi samples for quantitative applications

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The heterogeneity of ion signals is a severe problem in MALDI, which results in poor data reproducibility and makes MALDI unsuitable for quantitative analysis. By regulating sample plate temperature during sample preparation, thermal-induced hydrodynamic flows inside droplets of sample solution are able to reduce the heterogeneity problem. A room-temperature sample preparation chamber equipped with a temperature-regulated copper base block that holds MALDI sample plates facilitates precise control of the sample drying condition. After drying of sample droplets, the temperature of sample plates is returned to room temperature and removed from the chamber for subsequent mass spectrometric analysis. The areas of samples are examined with MALDI-imaging mass spectrometry to obtain the spatial distribution of all components in the sample. In comparison with the conventional dried-droplet method that prepares samples under ambient conditions without temperature control, the samples prepared with the method demonstrated herein show significantly better spatial distribution and signal intensity. According to observations using carbohydrate and peptide samples, decreasing substrate temperature while maintaining the surroundings at ambient temperature during the drying process can effectively reduce the heterogeneity of ion signals. This method is generally applicable to various combinations of samples and matrices.

Parlak alan görüntüleri ve örnek plakası 5 sıcaklığı ve 25 ° C ile hazırlanan maltotrioz ve bradikinin fragmanı (1-7) MS Fotoğraflar, Şekil 1 &#39;de gösterilmiştir. Sodiated maltotrioz, iyon sinyali esas olarak doldurulur halinde örnek alanın çevresine 25 ° C lik bir örnek plakası sıcaklığı hazırlandığında. 5 ° C&#39;ye örnek plaka sıcaklığı düşürerek, sinyal homojen tüm numune alanı üzerinde doldurur. 5 ° C&#39;nin altında ö...

Watch this Scientific Journal Video about Preparation of Homogeneous MALDI Samples for Quantitative Applications at JoVE.com

Preparation of Homogeneous MALDI Samples for Quantitative Applications

Örnek Kurutma işlemleri esnasında alt-tabaka sıcaklığının ayarlanması sayesinde yöntemde MALDI kütle spektrometresi iyon sinyallerin uzaysal farklılıklarını azaltmak için bir kuralı gösterilmektedir.

Kantitatif Uygulamalar için Homojen MALDI Örneklerin Hazırlanması

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization ...

preparation-of-homogeneous-maldi-samples-for-quantitative-applications

Research

JoVE Journal

Biochemistry

8.7K Görüntüleme.  Academia Sinica.  Örnek Kurutma işlemleri esnasında alt-tabaka sıcaklığının ayarlanması sayesinde yöntemde MALDI kütle spektrometresi iyon sinyallerin uzaysal farklılıklarını azaltmak için bir kuralı gösterilmektedir. 

Video: Preparation of Homogeneous MALDI Samples for Quantitative Applications

Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. 13C labelling).
Electrospray ionization (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra.
Here we present an accessible approach for analyzing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and &#945;-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analyzing intact proteins larger than 100 kDa by MALDI-MS.

Matrix-assisted Laser Desorption/Ionization Time of Flight MALDI-TOF Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Accurate mass measurement represents an important step during the investigation of proteins. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) can be used for such determination and its key advantage is the tolerance to salts, detergents and contaminants. Here we illustrate an accessible approach for the analysis of proteins larger than 100 kDa by MALDI-MS.

Uçuş lazer desorpsiyon / iyonizasyon zaman sağlam proteinler (MALDI-TOF) kütle spektrometrik analizi Matris destekli daha büyük 100 kDa

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass ...

Kimya

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI Numune Hazırlama: Ultra İnce Tabaka Yöntemi

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Biyoloji

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

Biyomarker Discovery için Mezogözenekli Silika İnce Filmler üzerine düşük molekül ağırlıklı Protein Zenginleştirme

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Biyomühendislik

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

Örnekleri için matris peptit esaslı tamir Formalin en iyi hazırlık lazer desorpsiyon/iyonlaşma kütle spektrometresi iş akışlarını görüntüleme destekli

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

Biyokimya

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer's and Parkinson's disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma - mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.

Size exclusion chromatography hyphenated with inductively coupled plasma - mass spectrometry (ICP-MS) is a powerful tool to measure changes in the abundance of metalloproteins directly from biological samples. Here we describe a set of metalloprotein standards used to estimate molecular mass and the amount of metal associated with unknown proteins.

Boyut Dışlama ICP-MS kullanarak Nicel Metalloproteomic Analiz Standartları

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of ...

Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and biomarkers. Recently, it has been used in microbiology for the routine identification of bacteria grown from clinical samples, without preparation or fractionation steps. We and others have applied this whole-cell MALDI-TOF mass spectrometry technique successfully to eukaryotic cells. Current applications range from cell type identification to quality control assessment of cell culture and diagnostic applications. Here, we describe its use to explore the various polarization phenotypes of macrophages in response to cytokines or heat-killed bacteria. It allowed the identification of macrophage-specific fingerprints that are representative of the diversity of proteomic responses of macrophages. This application illustrates the accuracy and simplicity of the method. The protocol we described here may be useful for studying the immune host response in pathological conditions or may be extended to wider diagnostic applications.

This protocol describes the use of whole-cell MALDI-TOF mass spectrometry on eukaryotic cells. Here, we illustrate the accuracy of this technique by analyzing the multiple activation states of macrophages in response to their microenvironment.

Tüm hücre MALDI-TOF Kütle Spektrometre Makrofaj Aktivasyon Farklı Modu analiz için Doğru ve Hızlı Yöntem

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and ...

İmmünoloji ve Enfeksiyon

An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) MS is the method of choice in carbohydrate analysis, poor ion signal and data reproducibility of carbohydrate samples continue to be severe problems. For quantitative analysis of carbohydrates, an effective analytical protocol providing superior data quality is necessary. This video demonstrates sample preparation protocols to improve signal intensity and minimize data variation of carbohydrates in MALDI-MS. After drying and crystallization of sample droplets, the crystal morphology is reformed by methanol before mass spectrometric analysis. The enhancement in carbohydrate signal is examined with MALDI imaging mass spectrometry (IMS). Experimental results show that crystal reformation adjusts crystalline structures and redistributes carbohydrate analytes. In comparison with the dried droplet preparation method in conventional MALDI-MS, reforming carbohydrate crystal morphologies with methanol shows significantly better signal intensity, ion image distribution, and data stability. Since the protocols demonstrated herein do not involve changes in sample composition, they are generally applicable to various carbohydrates and matrixes.

A protocol for enhancing carbohydrate ion signals in MALDI mass spectrometry by reforming crystalline structures during sample preparation processes is demonstrated.

Matris yardımlı Lazer Desorpsiyon/iyonlaşma kütle spektrometresi karbonhidrat iyon sinyalleri geliştirmek için bir verimli örnek hazırlama yöntemi

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) ...

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix&#160;assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has&#160;the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.

Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging of small molecules; protocols for the use of DT for the MALDI imaging of endogenous lipids on the surface of tissue sections&#160;by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.

Bir Fourier Matrix Yardımlı Lazer Dezorpsivon / İyonizasyon Görüntüleme için bir Matrix olarak ditranol İyon Cyclotron Rezonans Kütle Spektrometresi Dönüşümü

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied1. Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples1-5. This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Mass spectrometric imaging (MSI) is a powerful tool that can be used to discover and identify various chemical species in intact tissues, preserving the compounds in their native environments, which can provide new insights into biological processes. Herein a MSI method developed for the analysis of small molecules is described.&#160;

Metabolitlerdeki Soruşturma MALDI-Kütle Spektrometrik Görüntüleme<em&gt; Medicago truncatula</em&gt; Kök Nodüller

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the ...

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to investigate the biological activities during development and diseases. Metabolomics approaches&#160;are widely employed in the study of cancer, nutrition/diet, diabetes, and other physiological and pathological conditions involving metabolic processes. An advantageous tool that aids in metabolomic profiling advocated in this paper is matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Its ability to detect metabolites in situ without labeling, structural modifications, or other specialized reagents, such as those used in immunostaining, makes MALDI MSI a unique tool in advancing methodologies relevant in the field of metabolomics. An appropriate sample preparation process is critical to yield optimal results and will be the focus of this paper.

The goal of this protocol is to provide detailed guidance on the sample preparation when planning for experiments using MALDI MSI to maximize metabolic and molecular detection in biological samples.

MALDI Kütle Spektrometresi Görüntüleme Kullanarak Metabolik Profilleme için Örnek Hazırlama

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to ...