Articles

Comprehensive Screen of Acidic/Neutral/Basic Drugs from Urine and Plasma Using Micro-Prep™ HLB and MMCX Extraction Plates and Analysis on LC–MS/MS

SEC–MALS Analysis of Exosomes Using the Shodex OHpak SB-806 HQ

Characterization of Monoclonal Antibodies Using Native SEC–MS and Its Comparison to Denaturing RP–MS Technique

Liposomes and lipid nanoparticles are often used as nanocarriers to encapsulate fragile nucleic acids and hydrophobic or highly toxic drugs, and to safely deliver them to target tissue. During drug nanocarrier product and process development, as well as quality control, it is of great importance to monitor liposome size distributions accurately while also verifying drug encapsulation. FFF-MALS-DLS, consisting of field-flow fractionation (FFF) combined with multi-angle light scattering (MALS) and dynamic light scattering (DLS), is a powerful tool for characterizing the size, concentration, and structure of large nanoparticle ensembles.

Encapsulation might cause changes in liposomal dimensions, but that is not always the case and other effects could cause such changes. Therefore, a more sophisticated analysis is warranted than mere size. Here, we report the analyses of two liposome samples, one empty and one filled with drug, by means of an EclipseTM FFF system followed by a DAWN® MALS detector with embedded WyattQELSTM DLS module. The FFF separation method was optimized with the aid of Wyatt’s proprietary FFF simulation software. ASTRA software was used to collect and analyze the light scattering data to determine size and concentration (number density).

FFF separates particles according to hydrodynamic radius. Thanks to upstream separation, quantitative size distributions by FFF-MALS-DLS provide far more resolution and quantification than batch (unfractionated) DLS. Online DLS directly measures the hydrodynamic radius, 

, sequentially for each eluting size fraction, while MALS simultaneously measures the root-mean square radius, 

, provides important structural information: it can discriminate between empty and filled shells or quantify the axial ratio of a uniform ellipsoid.

 are plotted against elution time in Figure 1. The results from duplicate runs demonstrate clean separation and excellent reproducibility of the FFF-MALS-DLS method. Figure 1(a) shows that the 

 values for both empty and filled liposomes are well-overlaid, which is expected since FFF separates according to hydrodynamic size. However, as shown in Figure 1(b), 

 values for these two liposomes do not overlay, which indicates different internal structures.

 is the slope of the linear fit. Interpretation of 

 requires an assumption about the shape of the particles; here we make use of a priori knowledge that they are spherical. The values of 

 for these two populations then correlate precisely to empty and filled liposomal structures.

In addition to size and structure, FFF-MALS can determine quantitative size distributions (number density vs. size) of size-fractionated nanoparticles if the refractive index of the constituent material is known. For lipids this is quite straightforward and Figure 2(b) provides the quantitative nanoparticle concentration analysis.

For liposomes or other nanoparticles, FFF-MALS-QELS provides an easily adaptable yet powerful characterization tool to obtain information on particle size, size distribution, particle count, as well as structure—all without making assumptions about the particles or their composition. FFF-MALS-DLS instrumentation is essential for robust drug nanocarrier development and quality control.

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Wyatt – Liposome Size, Concentration, and Structural Characterization by FFF-MALS-DLS

The timsTOF Pro platform, introduced in 2017, already featured a sensitivity boost from the Parallel Accumulation Serial Fragmentation (PASEF®) technology, which provides time-and-space focusing of the ions in the Trapped Ion Mobility Spectrometry (TIMS) tunnel. The timsTOF SCP platform further enhances the sensitivity with robustly modified ion optic design. This boost in sensitivity enables measurement of low nanogram and sub-nanogram peptide loads, resulting in quantification of a few thousand protein groups per injection. Our data from as low as 200 pg of peptide loads demonstrates the applicability to unbiased true single cell proteomics in a routine fashion.

Single-cell ‘omics in recent years has highlighted the microheterogeneity in clonal populations. Unlike other ’omics technologies, single-cell proteomics is hindered by lack of amplification techniques for protein molecules. Single-cell proteomics is now beginning to get attention, and one common strategy is to multiplex labeled single cells together with carrier samples to boost the sensitivity in detection. Further improvements in sophisticated sample preparation techniques have resulted in increased peptide yield that is delivered to the liquid chromatography–mass spectrometry (LC–MS) instrumentation (1). While these improvements upstream of sample measurement have improved the analysis depth, the raw sensitivity requires improvement as well without compromising robustness. Parallel Accumulation and Serial Fragmention (PASEF) (2) on the timsTOF Pro platform makes efficient usage of the ion beam and with intelligent precursor placement within a TIMS cycle achieves rapid-sequencing speed. In addition, the ions get focused in space and time within the TIMS cell, resulting in a significant boost in sensitivity. This enables the analysis of low sample amounts, in the range of low-nanogram peptide loads. The newly designed timsTOF SCP’s ion optics allow a 4–5× improvement in ion current by increasing the ion brightness while maintaining the robustness of the timsTOF Pro. As the yield of the electrospray ionization increases with lower flow rates, we further enhanced the experiment’s overall sensitivity by coupling the timsTOF SCP to an Evosep One (Evosep Biosystems) instrument operated with the new low-flow Whisper methods. We have characterized the resulting system’s performance by injecting and measuring peptide loads mimicking the amount resulting from a single-cell preparation.

The new ion optic design of the timsTOF SCP system includes switching the orientation of the ion optics with inclusion of an additional ion funnel and additional orthogonal turns of the ion beam to preserve the robustness of the instrument. The source contained a wider glass capillary orifice that draws more ions into an additional funnel housed in a multi-stage differentially pumped region. Our initial experiments demonstrated that in addition to the brighter ion beam these dedicated modifications were crucial to gain a factor five boost in ion current. For ultrahigh sensitivity measurements from 200 pg to a few nanograms of peptide, we coupled an Evosep One system (Evosep Biosciences) to the timsTOF SCP instrument and used a ~28 min gradient Whiper 40SPD method that offers a constant flow of 100 nL/min. Evotips were loaded with K562 (Promega) peptides according to the vendor instructions. Data were acquired in a DIA mode with window placements as shown (Figure 1B). All data were processed using Spectronaut software version 14 with default settings applying a hybrid library.

A dilution series of peptide load was performed starting from 200 pg to 25.6 ng in replicates using the ultralow flow method—Whisper 40SPD— from Evosep Biosystems. This method delivers gradient at a flow rate of 100 nL/min further boosting the sensitivity of the platform. About 1200 protein groups could be quantified from the 200 pg loads, and that number increased to an excess of 4000 protein groups for 6.4 ng loads. Then 250 and 500 pg loads, mimicking the amount of peptides resulting from the digestion of one or two isolated cells, were used to test the accessible proteome depth. These samples were analyzed using the Whisper 40 samples per day (SPD) method applying dia-PASEF® methods with a 0.7 cycle time method that covers between 400 and 1000 

. The data were processed with a library consisting of 5200 protein groups and about 54,000 peptides. From 250 and 500 pg loads on average 1542 and 2146 protein groups were quantified, respectively.

Work done in the laboratories of Prof. Matthias Mann with the timsTOF SCP combined with robust low flow Evosep One when applied with efficient sample preparation yields exciting results on the biology of the cell cycle (3,4).

timsTOF SCP provides robust proteome coverage with peptide loads in the range of 250 pg.

Combination of timsTOF SCP with Whisper methods on the Evosep provide a robust and sensitive platform to perform single-cell proteomics.

(3) A-D Brunner et al., https://doi.org/10.1101/2020.12.22.423933 

(4). A. Mund et al., https://doi.org/10.1101/2021.01.25.427969

Bruker – Ultrahigh Sensitivity Proteomics on the timsTOF SCP

While not currently regulated, ultrashort-chain (C2–C3) per- and polyfluoroalkyl substances (PFAS) are of great interest. Current testing methodologies using reversed-phase liquid chromatography (LC) columns cannot be used because of a lack of retention, so either a separate method or a different column is required.

A unique, hybrid ion-exchange/HILIC column (Raptor Polar X) was used to develop a comprehensive LC–MS/MS method for the analysis of ultrashort-chain through long-chain, and alternative PFAS in water sources (tap, river, groundwater, and sewage effluent). The Raptor Polar X‘s multimode retention mechanisms allow for retention with a single isocratic run.

Chromatographic conditions are reported in Figure 1.

To avoid introducing background contamination, polypropylene vials and caps were used during sample preparation.

Each water sample of 250 μL was mixed with 250 μL of methanol and 5 μL of internal standard solution (10 ng/mL of 13C2-PFHxA, 13C2-PFOA, 13C3-PFBS, 13C4-PFOS in methanol).

Calibration standards were prepared by using deionized water and fortified with 14 analytes (see Figure 1) at a range of 10–800 ng/L. The calibration standard solutions were diluted 1:1 as above.

A Restek tap water sample, along with three water samples (river, ground, and sewage effluent) supplied by the United States Environmental Protection Agency were fortified at 40 and 160 ppt. Blank and fortified water samples were diluted 1:1 in methanol as above for chromatographic analysis. For TFA measurement in groundwater, the sample was diluted fivefold with deionized water before fortification due to its high TFA concentration.

All analytes were eluted in 4 min with good peak shapes (Figure 1). The overall analytical cycle time was 8 min to ensure no matrix-related interferences.

Method linearity from 20–800 ppt for trifluoroacetic acid (TFA) and 10–800 ppt for all other analytes provided 

 values >0.996 and deviations <20% using a 1/x weighted quadratic regression.

Samples were fortified at the low and high concentrations of their calibration ranges and run in duplicate for each analytical batch. A total of three batches were measured on different days. Concentrations of fortified samples were adjusted to account for any observed background contamination in sample blanks. Results are presented in Table I.

These results demonstrate that switching to a mixed-mode LC column provides the capability to analyze currently monitored and emerging PFAS contaminants in a single, short, isocratic run, preparing laboratories for the future of PFAS testing.

Comprehensive Analysis of C2–C8 PFAS Using a Novel LC Column

Mono and disaccharides are of increasing importance in the growing industrialized food industry. With the rise of diabetes and other metabolic diseases, nutritional content has found itself at the forefront of consumer concern. Investigation of the constituents found on the packaging is now commonplace. Consumer concern, coupled with the increase in FDA regulation on consumer products, has brought about a change in individual ingredients reported on labels. Identification of the five most common mono and disaccharides found in packaged foods, lactose, maltose, sucrose, fructose, and glucose, is easily resolved on any of the RCX-30 family of columns offered by Hamilton Company.

Ultraviolet absorption, normally the detection method of choice for most chromatography separations, is unsuitable for carbohydrates. This means other analyte detection methods must be used, such as refractive index (RI), conductivity, and pulsed amperometric detection (PAD). Both RI and conductivity are excellent choices for detection if only isocratic methods are needed. PAD, however, offers the advantage of detection from either gradient or isocratic elution. An additional consideration when choosing a detector is sensitivity. RI and conductivity provide nanomolar detection levels while the detection limit for PAD is an order of magnitude lower.

The RCX-30 columns, powered by a propyl trimethylamine functionality, use a strong anion exchange mechanism to aid carbohydrate separation. Increasing saccharide retention is easily achieved by increasing pH or decreasing hydroxide concentration.

Similarly, initiating the opposite operations facilitate a decrease in retention and allow a fine-tuning of the individual analytes. The PS-DVB particles featured in all Hamilton polymeric columns provide exceptional column lifetime and reduced backpressure. Polymeric columns always provide excellent pH stability in alkaline mobile-phase conditions because there are no silanols to degrade, offering consistent performance, injection after injection.