The authors acknowledge the Smart MAterials Design, Analysis, and Processing consortium (SMATDAP) funded by the National Science Foundation under cooperative agreement IIA-1430280 and the LA Board of Regents for a graduate fellowship (MEP). Polymer samples for these experiments were provided by MilliporeSigma (Sigma-Aldrich).&#160; Open Access publication of this article is sponsored by MilliporeSigma.

Caution: All reactions were run in a fume hood. Please read all Material Safety Data Sheets (MSDS) for any chemical used and take appropriate precautions.
1. Sample Preparation
<ol>
	<li>Preparation of the matrix stock solutions
	<ol>
		<li>Dissolve 20 mg of &#945;-cyano-4-hydroxycinnamic acid (HCCA) in 1 mL of tetrahydrofuran- unstabilized (THF), and vortex until dissolved.</li>
		<li>Dissolve 20 mg of 2,5-dihydroxybenzoic acid (DHB) in 1 mL of THF, and vortex until dissolved.</li>
	</ol>
	</li>
	<li>Preparation of the alkali cation stock solution
	<ol>
		<li>Dissolve 2 mg of sodium trifluoroacetate (NaTFA) in 1 mL of THF, and vortex until dissolved.</li>
		<li>Dissolve 2 mg of potassium trifluoroacetate (KTFA) in 1 mL of THF, and vortex until dissolved.</li>
	</ol>
	</li>
	<li>Preparation of analyte stock solutions
	<ol>
		<li>Sample 1: Dissolve 2 mg of poly(ethylene glycol) 2-aminoethyl ether acetic acid (Mn = 5000) in 0.5 mL of THF, and vortex until dissolved.</li>
		<li>Sample 2: Dissolve 2 mg of polyoxyethylene bis(azide) (Mn = 2000) in 0.5 mL of THF, and vortex until dissolved.</li>
		<li>Sample 3: Dissolve 2 mg of poly(L-lactide), thiol terminated (Mn = 2500) in 0.5 mL of THF, and vortex until dissolved.</li>
	</ol>
	</li>
	<li>Preparation of sample mixtures for analysis
	<ol>
		<li>Prepare a series of solutions by mixing matrix, analyte and cation solution while varying the relative proportions of the components such that nine unique samples mixtures are made. For example, keeping the amount of added matrix stock solution constant (e.g., 10 &#181;L), vary the amount of analyte solution by a factor of three (e.g., 45, 15, and 5 &#181;L), while also varying the amount of cation solution by a factor of three (e.g., 9, 3, and 1 &#181;L). These samples effectively yield a 3 x 3 grid of samples with the two different concentration variables on the x and y axes (Figure 1).</li>
		<li>For the representative example, combine 15 &#181;L of the poly(L-lactide) solution, with 15 &#181;L of the DHB solution, and 1 &#181;L of the NaTFA solution.</li>
		<li>Pipette 1 &#956;L of each solution mixture onto an individual sample well on the MALDI target plate (Figure 2). Add the samples incrementally in small portions to prevent the sample from flowing out of the sample well, allowing each aliquot to evaporate to dryness before adding additional sample. 
		NOTE: For higher boiling point solvents, an air gun may be necessary to expedite solvent evaporation, though caution should be used to avoid heating the sample plate, which might cause the plate to warp.</li>
	</ol>
	</li>
	<li>Preparation of standard samples for calibration
	<ol>
		<li>Prepare the calibration standards using the suggested supplier protocol. 
		NOTE: Dendrimer calibrants were selected for this study and are available as the pure dendrimers or premixed with matrix, calibrant and cation at optimized ratios.</li>
	</ol>
	</li>
</ol>
2. Data Acquisition Optimization
<ol>
	<li>Initiating Data Acquisition
	<ol>
		<li>Open data acquisition software &#34;FlexControl&#34;.</li>
		<li>Eject the platform to enable the loading of the target plate by pressing the &#34;<img alt="Equation 1" src="/files/ftp_upload/57174/57174eq1v2.jpg" />&#34; button.</li>
		<li>Gently place the target plate with the loaded calibrant and analyte samples on the platform in the appropriate orientation.</li>
		<li>Use the acquisition software to inject the target plate on the platform by pressing the &#34;<img alt="Equation 1" src="/files/ftp_upload/57174/57174eq1v2.jpg" />&#34; button again.</li>
		<li>Select an appropriate data acquisition method (positive mode acquisition method) by pressing File | Select Method. 
		NOTE: For most polymer samples, including our representative analytes, ionization is expected via complexation with a cation and, therefore, a positive mode acquisition method is most appropriate. Depending on the instrument, for lower mass ranges (500-10,000 Da) or when higher resolution is desired, select a reflector mode method file. For higher molecular weight samples or when higher signal sensitivity is needed, and lower resolution is acceptable, select a linear mode method file.</li>
		<li>Before acquiring data, make sure an appropriate mass range for collecting data is selected-ideally the mass range will include half of the lowest mass in the expected distribution as well as double the highest mass in the expected distribution. Check this by clicking on the Detection tab and viewing Mass Range. 
		NOTE: This helps to ensure that signal from lower molecular weight degradation fragments or higher molecular weight aggregates (dimer) that may be present in the sample are included in the data set. Also, note that matrix oligomers are frequently noticed with high signal intensities in most MALDI-TOF mass spectra, providing high intensity noise with masses as high as 1,000 Da, complicating analysis below this mass. Although the calibration will be required before obtaining a final data set, an accurate calibration file can only be acquired if identical acquisition parameters are used as those optimized for the particular analyte. Therefore, a preliminary optimization of the analyte mass spectrum is required before the calibration, followed by the reacquisition of a calibrated analyte mass spectrum.</li>
	</ol>
	</li>
	<li>Preliminary data acquisition
	<ol>
		<li>From the acquisition software, select the position on the target plate which corresponds to the desired analyte.</li>
		<li>Initiate collecting data while moving the laser target around the sample to maximize the signal. To initiate data collection, press Start. 
		NOTE: The laser can exhaust matrix at a particular location after repeated sampling.</li>
		<li>Using the slide bar on the left side of the camera window, adjust the laser power such that the minimum power necessary to achieve isotopic resolution is achieved. 
		NOTE: When analyzing multiple samples to confirm the optimal ratio of analyte, cation, and matrix, use the same laser power on each of the analyte samples to determine which sample exhibits the best signal/noise ratio for those acquisition parameters. Continue future acquisition optimization with the sample that appears to exhibit the best signal to noise ratio.</li>
	</ol>
	</li>
	<li>Data acquisition optimization
	<ol>
		<li>Zooming in on an individual peak in the middle of the mass range of interest, optimize the resolution by adjusting the difference in acceleration voltages (for the instruments in this study, this involves adjusting the &#34;IS2&#34; value), which is in the Spectrometer tab. 
		NOTE: This is most rapidly optimized by varying the IS2 value in large steps, taking note of which value generates the best resolution (i.e., the smallest full peak width at half maximum signal intensity), and then further optimizing in smaller steps of the IS2 value. Typically, the optimal IS2 value is higher (closer to IS1) for low mass polymers, and lower for high mass polymers.</li>
		<li>If desired, increase resolution using reflectron mode. 
		NOTE: Reflectron mode allows for the compensation of discrepancies in initial velocity of ions of the same m/z by forcing the higher velocity ions of the same m/z value into a longer path to the detector. This increase in path to the detector allows for slower ions of the same m/z value to arrive at the detector simultaneously, effectively focusing the ions for increased resolution. Although reflectron mode typically improves the signal resolution, for samples with weak signal intensity, linear mode might be required in order to visualize the data.</li>
		<li>Finally, optimize the laser power by reducing the laser power as low as possible while still generating a reasonable signal to noise ratio (e.g., signal to noise ratio of approximately 10). 
		NOTE: Because higher laser powers generally reduce the resolution and can induce fragmentation, the mass spectra with best quality are acquired using reduced laser power but a higher number of scans.</li>
		<li>Once the acquisition parameters are optimized, save the uncalibrated mass spectra by selecting File and then Save Spectrum to File As. For external calibration, a new acquisition of the calibrant under these identical, optimized parameters must be carried out before a new acquisition is initiated to generate the calibrated mass spectrum of the analyte.</li>
	</ol>
	</li>
</ol>
3. MALDI Calibration
<ol>
	<li>Acquisition of calibration mass spectrum
	<ol>
		<li>Using the acquisition parameters already optimized for the analyte, acquire an optimized mass spectrum of the sample of mass standards. 
		NOTE: Ideally, the calibration set should include one standard above the range of interest, one below, and at least one in the range of interest. The accuracy of the calibration is best if all acquisition parameters are identical for both samples.</li>
	</ol>
	</li>
	<li>Create a calibration file
	<ol>
		<li>Make sure that any existing calibration is voided or in a position to be overwritten by pressing Invalidate Calibration under the Spectrometer tab.</li>
		<li>Using the same acquisition parameters (e.g., laser power, IS2 voltage), move the laser over to the sample well containing the calibrant (e.g., dendrimer standard, peptide) by selecting the corresponding well with the cursor and acquire a spectrum by pressing Start.</li>
	</ol>
	</li>
	<li>Once sufficient signal has been acquired, press Start to finish acquiring data.</li>
	<li>Once a mass spectrum of the calibrant has been acquired, select the Mass Control List dropdown menu in the Calibration tab that corresponds to that calibration standard. The appropriate mass control list will have the reference masses of the calibrant selected with the appropriate cation. 
	NOTE: These should be available from the calibrant supplier, and make sure to use the monoisotopic mass values when isotopic resolution is achieved (e.g., reflector mode below m/z = 5000), and an average mass values when isotopic resolution cannot be achieved (e.g., linear mode above m/z = 5000),</li>
	<li>Before matching the corresponding reference peak to each selected calibrant peak, ensure that an appropriate peak picking protocol is being used by selecting the Processing tab. 
	NOTE: Peak picking protocols can vary based on spectral resolution. For an average mass calculation, the software should take the mass average across the entire series of isotopic peaks. For a monoisotopic mass calculation, the software should be set to calculate the exact mass of only the first isotopic peak.</li>
	<li>Apply the reference mass from the mass control list to the corresponding signal for the calibrant mass spectrum by selecting the area to the left of the peak of interest and then clicking on the corresponding mass in the control list to apply. Continue the process for the remaining calibrant peaks. 
	NOTE: For the most accurate and precise calibration, place the analyte and calibrant samples as close together as possible on the target plate, because subtle variations in target plate height can affect the accuracy of the calibration.</li>
	<li>Reacquire the analyte spectrum once the mass scale for the optimized acquisition parameters have been calibrated.</li>
</ol>
4. Data Analysis and Interpretation
<ol>
	<li>Peak picking
	<ol>
		<li>Open the analyte spectrum in the data analysis software (FlexAnalysis).</li>
		<li>Zoom in on a peak to identify if isotopic resolution has been achieved by selecting the Zoom in X-Range button.</li>
		<li>Press Mass List | Find to select peaks. If the monoisotopic peak is resolved, select this first peak in the isotopic distribution to determine its mass using a monoisotopic peak-picking protocol. If the monoisotopic peak is not resolved, use an average mass peak picking protocol, and determine the average mass of the entire isotopic distribution.</li>
		<li>Continue this peak picking process for each n-mer in the polymer distribution.</li>
	</ol>
	</li>
	<li>Polymer characterization and end group analysis calculations 
	NOTE: When used correctly, MALDI-TOF MS can provide valuable, accurate data for mass distribution calculations of polymers. It should be noted that mass distribution data is only accurate when the dispersity of the polymer sample is relatively low (e.g., approximately &#272;=1.3 or below).
	<ol>
		<li>Calculate the number average molecular weight, the mass average with respect to the number of moles of each mass fraction, using the formula: 
		<img alt="Equation 2" src="/files/ftp_upload/57174/57174eq2v2.jpg" /> 
		where Ni = number of molecules of a specific molecular weight and Mi = the specific molecular weight of those molecules.</li>
		<li>Calculate the weight average molecular weight, the mass average with respect to the weight of each mass fraction, using the formula: 
		<img alt="Equation 3" src="/files/ftp_upload/57174/57174eq3v2.jpg" /> 
		where N i = number of molecules of a specific molecular weight and M i = the specific molecular weight of those molecules.</li>
		<li>Once both the Mw and Mn have been calculated, quantify the breadth of the molecular weight distribution using the ratio Mw/Mn which is called dispersity, &#272;.</li>
		<li>The most unique and powerful feature of MALDI-TOF MS data analysis is the ability to determine or confirm the end groups of homopolymers. Determine the end group by rearranging the following formula for the observed mass of an n-mer in the mass spectrum (Mn-mer): 
		Mn-mer = n(MRU ) + MEG1 + MEG2 + Mion 
		where n = the degree of polymerization, 
		MEG1 = mass of the &#945;-end group, 
		MEG2 = mass of the &#969;-end group, 
		MRU = the mass of the repeat unit of the polymer, 
		and Mion = mass of the ion that complexes with the polymer.</li>
	</ol>
	</li>
</ol>

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</ol>

The authors have financial interest related to the SpheriCal calibrants used in this study.

MALDI-TOF mass spectrometry is an invaluable analytical tool for polymer characterization because of its ability to generate polymer ions in the singly charged state and with minimal fragmentation. This soft ionization technique utilizes short laser pulses to desorb solid-state samples of the polymer analyte embedded in a matrix compound to generate polymer ions in the gas phase. The macromolecules are typically ionized by complexation with cations that are added to the matrix to enable their analysis by mass spectrometry. These macromolecular ions are then accelerated by an extraction voltage to bring them into the field-free region of the flight tube which can enable their m/z to be determined based on their time-of-flight between the ion source and the detector5,32.
Compared to other polymer characterization techniques, MALDI-TOF MS spectra quality is heavily dependent on data acquisition parameters and sample preparation. Although there is no set formula for sample preparation, understanding the function of each component of the sample preparation allows for more rapid empirical optimization. The most important factor in MALDI sample preparation is selection of the matrix because compatibility of the matrix with the polymer analyte is critical for allowing excited matrix to generate single, desorbed macromolecules in an ionized state5,15,17,19. Once appropriate matrix and cationization agents have been selected, the correct ratio of analyte, matrix, and cationization agent must be determined. This can be achieved empirically by creating a two-dimensional grid of samples (Figure 1) on the MALDI-TOF MS target plate (Figure 2) with increasing matrix concentration on one axis and increasing cationization agent concentration on the other.
Similar to MALDI sample preparation, there is no set formula for determining data acquisition parameters; however, certain trends should be considered to expedite spectral optimization. Reflectron mode, which increases resolution but decreases overall signal, is typically chosen for lower mass ranges (in these examples, below 4,000 Da) where isotopic resolution can be achieved. In these cases, monoisotopic mass calculations and peak picking methods were used. For polymer samples with masses above 4,000 Da, linear mode was used with average mass calculations and peaking picking methods. To improve signal resolution, the ion source voltages should be adjusted in small increments with the general trend of larger mass polymers having a larger voltage differential (IS1 versus IS2).
While optimized sample preparation and acquisition parameters can provide precision, mass accuracy can only be achieved through effective calibration. The time-of-flight for a given mass can vary subtly with respect to the variable acquisition parameters and even plate positions, therefore a calibration should be carried out for each set of optimized acquisition parameters in order to yield accurate mass determinations5,30. Once the acquisition parameters and sample preparation have been optimized, the spectra should be calibrated using these exact same conditions.
Because of the exceptional resolution and mass accuracy observed in the optimized MALDI-TOF mass spectra of polymers, this technique has become a valuable complimentary tool for determining polymer mass distribution data. However, its ability to resolve individual repeat units within the polymer mass distribution provides a particular advantage for end group analysis relative to other polymer characterization techniques such as gel permeation chromatography (GPC) and nuclear magnetic resonance (NMR). This is particularly valuable for determining the fidelity of end group functionalization reactions and the quantitative nature of end group conjugations reactions. This manuscript has demonstrated the ability to resolve the mass of individual polymer repeat units with up to two decimals points of mass accuracy, enabling the confirmation of end group modifications with a high level of confidence. With the substantial advances that have been made recently in the field of precision polymer synthesis, MALDI-TOF MS is becoming an increasingly important tool for determine macromolecular structure and functionality.

With improvements in living polymerization techniques, precision polymers with quantitatively functionalized end groups are increasingly available1. The concurrent development of azide-alkyne and thiolene click chemistries has enabled the nearly quantitative coupling of macromolecules to other moieties, providing access to a range of hybrid materials2,3,4. However, precise analytical techniques are required to characterize both the starting materials and products of these polymer conjugation reactions. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) is a valuable soft ionization analytical technique for characterizing polymers because it can generate polymer ions in a single charge state with minimal fragmentation5,6. MALDI-TOF MS has major advantages over other conventional methods of polymer characterization because it can provide mass spectra with resolution of the individual n-mers within the polymer mass distribution. As a consequence, such mass spectra can provide precise information about the average molecular weight, repeat unit mass, and molecular weight dispersity7, which can in turn elucidate competing polymerization mechanisms such as chain transfer8. However, MALDI-TOF MS is particularly powerful at providing information about polymer end groups9,10, which can be used to confirm end group modifications10,11 and other transformations12 such as polymer cyclizations11,13. Equally important, the relatively small amount of analyte (sub-micrograms) required for mass spectrometric analysis makes this technique useful for characterization when only trace quantities of material are available.
The MALDI-TOF MS analysis of polymers can be divided into four distinct steps: sample preparation, instrument calibration, spectral acquisition, and data analysis. Sample preparation is the most essential step for generating optimized MALDI-TOF mass spectra and occurs before the sample is introduced into the instrument14,15. The selection of an appropriate matrix with similar solubility parameters as the polymer analyte is critical to obtain high quality MALDI-TOF mass spectra and guidelines for matrix selection have been reported elsewhere14,15,16,17. A database of polymer MALDI &#34;recipes&#34; for sample preparation has also been published online18. For novel polymers, matrix selection can be approached by first understanding the solubility of the polymer and selecting a matrix with similar solubility parameters14,19. Polymers with high proton affinity can be protonated by most matrices14 (which frequently contain carboxylic acid groups), but for other polymers, a cationization agent is required14. Alkali ions adduct well with oxygen-containing species (e.g. polyesters and polyethers), whereas unsaturated hydrocarbons (e.g. polystyrene) adduct with transition metals such as silver and copper ions14,19. Because the polymer samples in this experiment contained oxygen atoms in the backbone, sodium or potassium trifluoroacetate (TFA) were used as the cationization agent. Once the matrix and cationization agents have been selected, the relative proportions of analyte, cation agent, and matrix must be carefully optimized to ensure high signal to noise. In this procedure, the parameters for sample preparation have already been optimized, however an empirical sample optimization procedure (step 1.4.1., Figure 1) that systematically varies the concentrations of the three components (analyte, matrix and cation) is effective for rapidly determining their optimal ratios.
Data acquisition also requires the optimization of a number of parameters. The most important parameters include the positive or negative ion mode of the spectrometer, the instrument operation mode (linear versus reflector), the acceleration voltage, and the extraction delay time. Another way that resolution can be increased is through the utilization of &#34;reflectron&#34; mode20,21,22,23. Reflectron mode essentially doubles the flight path of the ions to the detector by reflecting the ions at the end of the flight tube back towards a detector near the source while refocusing ions with different momentums, and therefore increasing the resolution though decreasing signal strength. In addition, higher resolution spectra can be obtained by decreasing the laser power which minimizes the signal-to-noise ratio by decreasing the number and energy of collisions and therefore reducing the fragmentation and kinetic inhomogeneities24. By tuning all of these parameters, the ions can be focused to minimize the effect of any inhomogeneity in initial position or velocity that occurs during the laser desorption process. When the acquisition parameters are optimized, isotopic resolution can often be achieved for ions with masses in excess of 10,000 Da, though this is also dependent upon the length of the flight tube and the instrument design. Most organic compounds that contain at least one heteroatom are prone to complexing with alkali cations such as lithium, sodium, and potassium. Many of the alkali metals are monoisotopes or of limited isotopes and therefore do not broaden the distribution.
While the instrument parameters can be tuned to optimize data precision, data accuracy is only achieved with an appropriate calibration11. Proteins and peptides were originally used as calibrants due to their monodispersity and availability, but suffer from variable stability and the prevalence of impurities25. More cost-effective and stable alternatives have included inorganic clusters and polydisperse polymers26,27,28,29. Unfortunately, these alternatives feature disperse masses, which complicate mass assignments, as well as smaller masses overall, making them useful only for calibrations below 10,000 Da. To combat these issues, Grayson et al.25 developed a dendrimer-based, polyester MS calibration system that is monodisperse, and boasts both broad matrix and solvent compatibility, shelf-life stability (&#62; 8 years), and lower production cost. Based upon the strengths of this system, it was selected as the calibrant for these experiments.
There are two main types of calibration: internal and external30. When calibrating externally, a standard with masses that bracket that of the analyte are placed on the MALDI target plate in a different sample position than the analyte to generate a separate mass spectrum from which a calibration file can be generated. On the other hand, increased accuracy can often be achieved with an internal calibration, which involves mixing the calibrant with the analyte to obtain a hybrid spectrum with both calibrant and analyte signals. In the procedure described below, an external calibration was implemented. After proper calibration of the mass scale, accurate analyte mass data can be acquired. To ensure the most accurate calibration, it is important that the data acquisition occurs soon after the calibration.
Finally, once the optimized, calibrated data sets were acquired, and the data were analyzed to provide structural information about the polymer samples. The spacing between n-mers within the polymer distribution can provide accurate measurement of the repeating unit mass. The number average molecular weight (Mn) and other mass distribution calculations (e.g., Mw (weight average molecular weight) and &#272; (dispersity)) can also be determined from the signal distribution in the mass spectra (step 4.2 for calculations). Perhaps most uniquely, in the case of homopolymers, the sum of the end group masses can be confirmed by determining the offset of the polymer distribution with respect to the mass of the repeating units alone. The information-rich MALDI-TOF mass spectra provide valuable characterization data that are complementary to more traditional polymer characterization techniques such as size exclusion chromatography, Fourier-transform infrared spectroscopy, and nuclear magnetic resonance.

<table><tbody><tr><td>polyoxyethylene bis(azide) (Mn=2000)</td><td>MilliporeSigma (Aldrich)</td><td>689696</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/689696?lang=en&amp;amp;region=US</td></tr><tr><td>poly(ethylene glycol) 2-amino-ethyl ether acetic acid (Mn= 5000)</td><td>MilliporeSigma (Aldrich)</td><td>757918</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/757918?lang=en&amp;amp;region=US</td></tr><tr><td>poly(L-lactide), thiol terminated (Mn=2500)</td><td>MilliporeSigma (Aldrich)</td><td>747386</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/747386?lang=en&amp;amp;region=US</td></tr><tr><td>SpheriCal&amp;reg;&amp;nbsp; peptide low</td><td>MilliporeSigma (Sigma-Aldrich)</td><td>PFS20</td><td>http://www.sigmaaldrich.com/catalog/product/sial/pfs20?lang=en&amp;amp;region=US</td></tr><tr><td>SpheriCal&amp;reg;&amp;nbsp; peptide medium</td><td>MilliporeSigma (Sigma-Aldrich)</td><td>PFS21</td><td>http://www.sigmaaldrich.com/catalog/product/sial/pfs21?lang=en&amp;amp;region=US</td></tr><tr><td>SpheriCal&amp;reg;&amp;nbsp; peptide high</td><td>MilliporeSigma (Sigma-Aldrich)</td><td>PFS22</td><td>http://www.sigmaaldrich.com/catalog/product/sial/pfs22?lang=en&amp;amp;region=US</td></tr><tr><td>2,4 dinitrofluorobenzene</td><td>TCI</td><td>A5512</td><td></td></tr><tr><td>maleimide</td><td>MilliporeSigma (Aldrich)</td><td>129585</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/129585?lang=en&amp;amp;region=US</td></tr><tr><td>1-ethynylfluorobenzene&amp;nbsp;</td><td>Fisher Scientific</td><td>766-98-3</td><td></td></tr><tr><td>triethylamine</td><td>MilliporeSigma (Sigma-Aldrich)</td><td>471283</td><td>http://www.sigmaaldrich.com/catalog/product/sial/471283?lang=en&amp;amp;region=US</td></tr><tr><td>N,N,N&#039;,N&quot;,N&quot;-pentamethyldiethylenetriamine</td><td>MilliporeSigma (Aldrich)</td><td>369497</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/369497?lang=en&amp;amp;region=US</td></tr><tr><td>Copper(I)Bromide</td><td>MilliporeSigma (Aldrich)</td><td>254185</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/254185?lang=en&amp;amp;region=US</td></tr><tr><td>glacial acetic acid</td><td>Fisher Scientific</td><td>A38212</td><td></td></tr><tr><td>sodium metabisulfite</td><td>MilliporeSigma (Sigma-Aldrich)</td><td>13459</td><td>http://www.sigmaaldrich.com/catalog/product/sigald/13459?lang=en&amp;amp;region=US</td></tr><tr><td>potassium trifluoroacetate</td><td>MilliporeSigma (Aldrich)</td><td>281883</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/281883?lang=en&amp;amp;region=US</td></tr><tr><td>trans-2-[3-(tert-butylphenyl)-2-methyl-2-properylidene]malononitrile</td><td>MilliporeSigma (Aldrich)</td><td>727881</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/727881?lang=en&amp;amp;region=US</td></tr><tr><td>a-cyano-4-hydroxycinnamic acid</td><td>MilliporeSigma (Sigma)</td><td>C8982</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/c8982?lang=en&amp;amp;region=US</td></tr><tr><td>tetrahydrofuran</td><td>Fisher Scientific</td><td>T425-1</td><td></td></tr><tr><td>dichloromethane</td><td>VWR Analytical</td><td>BDH1113-4LG</td><td></td></tr></tbody></table>

characterization of synthetic polymers via matrix assisted laser desorption ionization time of flight (maldi-tof) mass spectrometry

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group analysis as matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). This tutorial demonstrates methods for optimization of the sample preparation, spectral acquisition, and data analysis of synthetic polymers using MALDI-TOF MS. Critical parameters during sample preparation include the selection of the matrix, identification of an appropriate cationization salt, and tuning the relative proportions of the matrix, cation, and analyte. The acquisition parameters, such as mode (linear or reflector), polarization (positive or negative), acceleration voltage, and delay time, are also important. Given some knowledge of the chemistry involved to synthesize the polymer and optimizing both the data acquisition parameters and the sample preparation conditions, spectra should be obtained with sufficient resolution and mass accuracy to enable the unambiguous determination of the end groups of most homopolymers (masses below 10,000) in addition to the repeat unit mass and the overall molecular weight distribution. Though demonstrated on a limited set of polymers, these general techniques are applicable to a much wider range of synthetic polymers for determining mass distributions, though end group determination is only possible for homopolymers with narrow dispersity.

Watch this Scientific Journal Video about Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry at JoVE.com

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry

A protocol for the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) characterization of synthetic polymers is described including the optimization of sample preparation, spectral acquisition, and data analysis.

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group ...

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Organic Chemistry

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Unit 1

Synthetic Polymers

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25.2K Views.  Tulane University. A protocol for the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) characterization of synthetic polymers is described including the optimization of sample preparation, spectral acquisition, and data analysis.

Video: Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) is described. DNA extraction is carried out using a silica membrane-based DNA extraction kit. After the PCR amplification using a detergent-free PCR buffer, restriction enzymes are added to the solution without purifying the reaction solution. A reverse-phase silica monolith column and a Fourier transform high resolution mass spectrometer having a modified Kingdon trap analyzer are employed for separation and detection, respectively. The mobile phase, consisting of 400 mM 1,1,1,3,3,3-hexafluoro-2-propanol, 15 mM triethylamine (pH 7.9) and methanol, is delivered at a flow rate of 0.4 ml/min. The cycle time for LC/ESI-MS analysis is 8 min including equilibration of the column. Deconvolution software having an isotope distribution model of the oligonucleotide is used to calculate the corresponding monoisotopic mass from the mass spectrum. For analysis of oligonucleotides (range 26-79 nucleotides), mass accuracy was 0.62 &#177; 0.74 ppm (n = 280) and excellent accuracy and precision were sustained for 180 hr without use of a lock mass standard.

An improved polymerase chain reaction-restriction fragment length polymorphism method for genotyping pufferfish species by liquid chromatography/mass spectrometry is described. A reverse-phase silica monolith column is employed for separating digested amplicons. This method can elucidate the monoisotopic masses of oligonucleotides, which is useful for identifying base composition.

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by ...

Genetics

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.

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

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.

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

Bioengineering

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques. The sample peptoid is a nonamer consisting of alternating N-(2-methyloxyethyl)glycine (Nme) and N-(2-phenylethyl)glycine (Npe), as well as an N-(2-aminoethyl)glycine (Nae) at the N-terminus. The sequence formula of the peptoid is Ac-Nae-(Npe-Nme)4-NH2, where Ac is the acetyl group. The synthesis takes place in a commercially available solid-phase reaction vessel. The rink amide resin is used as the solid support to yield the peptoid with an amide group at the C-terminus. The resulting peptoid product is subjected to sequence analysis using a triple-quadrupole mass spectrometer coupled to an electrospray ionization source. The MS/MS measurement produces a spectrum of fragment ions resulting from the dissociation of charged peptoid. The fragment ions are sorted out based on the values of their mass-to-charge ratio (m/z). The m/z values of the fragment ions are compared against the nominal masses of theoretically predicted fragment ions, according to the scheme of peptoid fragmentation. The analysis generates a fragmentation pattern of the charged peptoid. The fragmentation pattern is correlated to the monomer sequence of the neutral peptoid. In this regard, MS analysis reads out the sequence information of the peptoids.

A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

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

Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass spectrometry (MS) is a popular tool but is not always sufficient to completely unveil all structural features. For example, although carbohydrates are biologically relevant, their characterization is complicated by numerous levels of isomerism. Ion mobility spectrometry (IMS) is an interesting complement because it is sensitive to ion conformations and, thus, to isomerism. 
Furthermore, recent advances have significantly improved the technique: the last generation of Cyclic IMS instruments offers additional capabilities compared to linear IMS instruments, such as an increased resolving power or the possibility to perform tandem ion mobility (IMS/IMS) experiments. During IMS/IMS, an ion is selected based on its ion mobility, fragmented, and reanalyzed to obtain ion mobility information about its fragments. Recent work showed that the mobility profiles of the fragments contained in such IMS/IMS data can act as a fingerprint of a particular glycan and can be used in a molecular networking strategy to organize glycomics datasets in a structurally relevant way. 
The goal of this protocol is thus to describe how to generate IMS/IMS data, from sample preparation to the final Collision Cross Section (CCS) calibration of the ion mobility dimension that yields reproducible spectra. Taking the example of one representative glycan, this protocol will show how to build an IMS/IMS control sequence on a Cyclic IMS instrument, how to account for this control sequence to translate IMS arrival time into drift time (i.e., the effective separation time applied to the ions), and how to extract the relevant mobility information from the raw data. This protocol is designed to clearly explain the critical points of an IMS/IMS experiment and thus help new Cyclic IMS users perform straightforward and reproducible acquisitions.

Ion mobility spectrometry (IMS) is an interesting complement to mass spectrometry for the characterization of biomolecules, notably because it is sensitive to isomerism. This protocol describes a tandem IMS (IMS/IMS) experiment, which allows the isolation of a molecule and the generation of the mobility profiles of its fragments.

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass ...

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.

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

Biology

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;

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

Introduction to Mass Spectrometry

Source: Laboratory of Dr. Khuloud Al-Jamal - King&#39;s College London

Mass spectrometry is an analytical chemistry technique that enables the identification of unknown compounds within a sample, the quantification of known materials, the determination of the structure, and chemical properties of different molecules.
A mass spectrometer is composed of an ionization source, an analyzer,&#160;and a detector. The process involves the ionization of chemical compounds to generate ions. When using inductively coupled plasma (ICP), samples containing elements of interest are introduced into argon plasma as aerosol droplets. The plasma dries the aerosol, dissociates the molecules, and then removes an electron from the components to be detected by the mass spectrometer. Other ionization methods such as electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) are used to analyze biological samples. Following the ionization procedure, ions are separated in the mass spectrometer according to their mass-to-charge ratio (m/z), and the relative abundance of each ion type is measured. Finally, the detector commonly consists in an electron multiplier where the collision of ions with a charged anode leads to a cascade of increasing number of electrons, which can be detected by an electrical circuit connected to a computer.
In this video, the procedure of ICP-MS analysis will be described by the detection of 56Fe as an example.

Mass Spectrometer: Principle, Operation, Applications

Source: Laboratory of Dr. Khuloud Al-Jamal - King&#39;s College London

Mass spectrometry is an analytical chemistry technique that enables the ...

JoVE Science Education

Analytical Chemistry

Matrix-Assisted Laser Desorption Ionization (MALDI)

Matrix-assisted laser desorption ionization (MALDI) is a powerful analytical technique used in mass spectrometry. It enables the identification and characterization of various biomolecules, including proteins, peptides, nucleic acids, and carbohydrates. MALDI spectrometry is widely employed in biological and medical research, as well as in fields like pharmacology and biochemistry.
The analyte of interest, a biomolecule or a mixture of biomolecules, is mixed with a suitable matrix material. The matrix is usually an organic compound, such as a small organic acid or a crystalline substance, that facilitates ionization. This analyte-matrix mixture is then deposited onto a target surface, inserted into the mass spectrometer, and dried. During drying, the matrix forms small crystals that incorporate the analyte molecules within their structure.
Once the sample is prepared, a pulsed laser is directed onto the target surface. The matrix molecules absorb the laser beam energy, causing them to heat up and vaporize rapidly. This process is known as laser desorption. As the matrix molecules vaporize, they carry the embedded analyte molecules. The vaporized analyte molecules become ionized through a process called proton transfer. The proton transfer occurs between the matrix and the analyte molecule. The matrix molecule donates a proton to the analyte molecule, resulting in the formation of protonated ions. The analyte ions are then accelerated by an electric field and further separated based on their mass-to-charge ratio in a time-of-flight mass analyzer.
The mass spectrum obtained from MALDI spectrometry provides valuable information about the mass and relative abundance of the analyzed biomolecules.

Desorpcja laserowa wspomagana matrycą (MALDI)

Matrix-assisted laser desorption ionization (MALDI) is a powerful analytical technique used in mass spectrometry. It enables the identification and ...

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