Summary

• The Intact Mass workflow in Byos® can deconvolve multiple types of molecules including proteins, peptides, and oligonucleotides, across a broad mass range.
• This application note outlines the setup of the Intact Mass Workflow for the measurement of isotopically resolved proteins and peptides with a molecular weight of less than 10,000 kDa.
• Statherin, a 5,379.6 Da salivary protein, was extracted from buccal swabs on a liquid handling system and successfully measured using the settings described here.

Introduction

The Intact Mass Workflow within Byos is capable of deconvolving a wide range of molecule types including proteins, peptides, and oligonucleotides. The charge deconvolution algorithm is also uniquely capable of handling wide molecular weight (MW) ranges derived from native mass spectrometer (MS) experiments, intact or reduced antibodies, as well as low molecular weight peptides or proteins which are isotopically resolved.

This application note describes how the Intact Mass Workflow can be set up to accurately measure isotopically resolved proteins or peptides less than 10,000 kDa. An example dataset will then be shown using the salivary protein statherin, one of several proteins which aid in the supersaturation and stabilization of oral calcium. Statherin was extracted using two different protocols on an Agilent AssayMap Bravo liquid handler and measured on a Thermo Scientific™ Q-Exactive™ MS. While this application note will use statherin (Figure 1) as an example protein, the workflow settings described would be appropriate for similarly sized peptide pharmaceuticals such as Liraglutide (3,751 Da), insulin (5,734 Da) and various pharmaceutical analogues, as well as smaller cyclic antimicrobial peptides such as Telavancin (1,755.6 Da).

 

Figure 1. Statherin sequence with phosphoserine S2, S3 sites. 

Experimental

Sample Collection and Preparation:

Saliva samples were collected from volunteers who provided informed consent prior to collection. Collection was carried out using cotton buccal swabs. Swabs were then suspended in 500 µL of PBS buffer, incubated at 300 RPM for 30 minutes at room temperature, then centrifuged at 10,000 x g for 10 minutes to pellet cellular and other insoluble materials. 125 µL of the supernatant was transferred to 96-well plates for extraction. Statherin extraction was carried out using two approaches on an Agilent AssayMap Bravo liquid handling system. First by C18 reverse phase SPE and secondly by immunopurification using biotinylated anti-statherin polyclonal antibodies immobilized on streptavidin cartridges (Figure 2).

Figure 2. Schematic of sample preparation workflow.

 

Mass Spectrometry and Liquid Chromatography:

Data were acquired with chromatographic parameters outlined below on a Q-Exactive system operating in positive full MS mode (Table 1).

Table 1.Sample preparation and acquisition parameters.

 

Data Analysis:

Acquired data were analyzed using Byos v5.3 with workflow settings described below. Custom Slices was used to set the trace integration range between 2.75 and 2.95 minutes as the elution time for the peptide was known (Figure 3).

Figure 3. Trace peak integration settings. Custom slices was used with an integration range between 2.75 and 2.95 minutes.

 

The statherin sequence DSSEEKFLRRIGRFGYGYGPYQPVPEQPLYPQPYQPQYQQYTF was entered into the chains table to calculate the reference mass for the protein (5216.5097 Da) and the doubly phosphorylated variant with sodium or potassium adducts were set as delta masses. In Byos v5.3, monoisotopic mass matching (vs Average) was introduced for more accurate mass matching for smaller molecules with sufficient resolution. Monoisotopic mass matching which was enabled with a tolerance of 10 ppm. Under the basic intact options, the neutral mass (3,000-7,000 Th) and m/z range (900 – 1900) was set in accordance with the molecule mass and acquisition settings (Figure 4).

 

Figure 4. Basic intact options and monoisotopic mass matching enabled. 

 

Under the advanced intact options, several adjustments were made from the default intact settings to properly correct the deconvolution algorithm to be compatible with low mass high resolution datasets (Figure 5).

Figure 5. Advanced intact options. 

 

            Several advanced commands were utilized to retain and render the isotopically resolved spectra as well as assist the algorithm to accept and compute masses with few charge states (Figure 6).

 

[Intact]

AdvancedMonoCalculation = true

MaxMonoisotope = 20000

ShowObservedMono = true

ChargeEnvelopeWeights = 0.1, 0.8, 0.1

 

*Note the MaxMonoisotope = 20000 will need to be adjusted for larger peptides

 

Figure 6. Advanced intact options. 

Results

Buccal swabs were collected from four individual donors to verify the consistency of detection across both C18 microextraction and immunoaffinity purification protocols as well as to test the analytical and deconvolution settings. Prior to analysis, the cotton portion of the swab was removed for the dowel, resuspended in PBS buffer, centrifuged at 10,000 x g to pellet cellular and insoluble material, then the supernatant used for statherin extraction as described above.

Statherin was positively identified in all the samples assessed. Intensities in the C18 extracted samples were stronger on average (avg 6.17E+06) compared to those prepared using affinity purification (avg 3.55E+06). However, the proportion of non-target background ions was significantly higher in the samples using the C18 cartridges as shown below in figure 7.

 

Figure 7. MS1 (left), Neutral mass (center), and zoomed in isotopic profile (right) for a saliva sample extracted via C18 microextraction (top) and immunopurification (bottom). 

 

         Reports generated in Byos can quickly assist with data analysis. Including relative intensity tables and barcharts as well as ppm error tables (Tables 2A-C).

 

Tables 2A-C. Expected mass intensity in tabular form (A), expected mass as an intensity based bar chart (B), and ppm error (C) across eight samples assayed.  

Conclusion

The workflow settings described here enable the deconvolution of isotopically resolved low MW peptides and proteins. Additionally, with monoisotopic mass matching (vs. Average) enabled in Byos v5.3, peaks can be assigned with more confidence than previous software versions for this type of molecule. The salivary protein statherin, with a molecular weight of 5,739.6 Da, was used an example protein. Statherin was extracted on an Agilent Bravo liquid handler using two techniques including reverse phase microextraction with C18 columns as well as affinity purification with immobilized anti-statherin polyclonal antibodies. Statherin was successfully identified in samples collected from four unique donors.