Biomarkers and omics

Separation and Analysis of Intact Prostate Specific Antigen(PSA) and its Proteoforms by CESI-MS Under Nativeand Denaturing Conditions.
• The ability to separate, ionize and detect intact PSA using the CESI 8000 Plus High Performance
Separation-ESI Module coupled to high mass resolution mass spectrometry under nativeand denaturing conditions• Improved ionization efficiency of PSA results in the identification of almost 2-fold greater PSA
proteoforms detectedMarcia R. Santos,1 Chitra K. Ratnayake,1 David M. Horn,2 Barry L. Karger,3 Alexander R. Ivanov,3 and Rosa Viner2
1 SCIEX, Brea, CA
2 Thermo Fisher Scientific, San Jose, CA
3 Northeastern University, Barnett Institute of Chemical and Biological Analysis, Boston, MAOverview
Despite recent advances in intact protein mass spectrometry(MS), challenges around detection and analysis of thesemolecules remain. Currently, most studies are performed byinfusion of highly purified proteins or by immunoaffinity analysis.
This is mainly due to the lack of high-efficiency front-end
separation tools that would allow for direct analysis of thesemolecules. Capillary electrophoresis (CE) is an established andpowerful technique for intact protein analysis, particularly in thecharacterization of biologics under both native and denaturingconditions. The integration of CE and ESI into a single dynamic
process within the same device (deemed CESI) provides

conditions resulted in the identification and quantification of 56
glycoforms. The total ion electropherograms acquired by CESIMS performed under denaturing and native conditions resultedin the detection of only 4–5 predominant peaks. However,the deconvolution analysis of the CESI-MS data under native
conditions revealed a possible 236 proteoforms in contrast
to only 127 identified by direct infusion. Due to the increased
signal-to-noise ratio under native conditions, we were able toidentify additional proteoforms compared to the analysis underdenaturing conditions. These hybrid proteoforms migrated
noticeably later than the sialylated/complex glycan species.

the means to perform highly efficient protein separation andIntroduction

ionization in the ultra-low nanoflow regime (~25 nL/min)

Prostate specific antigen (PSA) is a ~30 kDa glycoprotein,simply using an open capillary tube. Thus CESI-MS is ideallysuited for intact protein analysis under native or denaturingconditions by MS.
In this study, we used a neutral surface capillary to enable theseparation of proteoforms of PSA under both native anddenaturing conditions, aiming to identify and quantify the numberof proteoforms that can be detected in comparison with direct
infusion by nanoLC. The top 20 PSA proteoforms were identicalin experiments performed under both native and denaturingconditions, where HexNac4Hex5dHex1NeuAc2 was identified as
the most predominant glycan form, agreeing nicely with an ABRF
Interlaboratory study from 2012.1 The combination of infusion2and CESI-MS experiments using both native and denaturingcontaining 1 N-glycosylation site at Asn69 that is secreted by
the epithelial cells of the prostate gland. Despite the difficulties
in validating antibodies and their possible cross-reactivity, thetraditional and most robust method to detect and quantify protein
biomarkers has been immunoaffinity analysis. In this work, wedemonstrate a simple and fast method for the profiling of PSA
proteoforms under native or denaturing conditions using CESIMS. CESI-MS operates at ultra-low nL/min flow rates offering
several advantages, including increased ionization efficiency anda reduction in ion suppression (Figure 1A and Figure 1B).
In this study, we have detected more than 200 PSA proteoforms,
using CESI coupled with high-sensitivity, high-resolution mass
spectrometry compared to 127 proteoforms identified bynanoLC infusion.
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Materials and methods
Sample preparation:

Plus System connected to the Thermo Scientific Nanospray IIion source. Both types of separations (native and denaturing)were performed using a neutral coated capillary. For theHuman PSA sample was purchased from Lee Biosolutions (St.

background electrolytes, 10% acetic acid (pH 2.2) and 40 mMLouis, MO) and diluted to 400 ng/μl in 10 mM ammonium acetate

ammonium acetate (pH 7.5) were used for denaturing and native(pH 7.5) for native MS and into 10% acetic acid for all otherconditions, respectively. The data files were analyzed using

experiments.

Thermo Fisher Scientific Deconvolution 4.0 software.

CESI-MS conditions
Intact PSA was separated using a CESI 8000 Plus High
Performance Separation-ESI Module (SCIEX) equipped with aneutral coated OptiMS capillary operating in an ultra-low flowregime (Figure 1A). The CESI 8000 Plus System was coupled
to an LTQ Orbitrap Elite mass spectrometer (Thermo FisherScientific) configured for 120 K resolution at m/z 400. Intact PSA
under native conditions was analyzed on an Exactive Plus EMR
system (Thermo Fisher Scientific) at 140,000 FWHM resolution atm/z 200. Figure 1B shows the OptiMS sprayer of the CESI 8000

Important:• A separation current above 5 μA might cause permanent
damage to the separation capillary.
• Generally, please do not apply >2000V to generate
electrospray as it may result in capillary damage.

Direct infusion under native conditions
Using the Advion nanochip, 8 ng of PSA solution was directly
infused into an Exactive Plus EMR instrument at 20 nL/min.

Figure 1A. Schematic representation of the CESI 8000 Plus System with an OptiMS sprayer and adapter.

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Figure 2. (A) Base ion electropherogram of PSA under denaturingconditions showing the 4 resolved peaks. (B) Corresponding iondensity map demonstrates the CE migration and m/z patternsof the detected proteoforms.

Figure 1B. The OptiMS sprayer connected to a Thermo ScientificNanospray II ion source.

Results
CESI-MS under denaturing conditions
Separation of 4 predominant resolved peaks was achieved withdenatured PSA in 10% acetic acid (pH 2.2) using CESI-MS(Figure 2A). A corresponding ion density map illustrates thecomplexity of each peak (Figure 2B). The amount of PSA injectedin this experiment was 4.2 ng, which represents only 1.6% of thetotal capillary volume. However, the corresponding ion density

Figure 3. PSA profiling under denaturing conditions (panel A) andcorresponding deconvoluted spectra (panel B).

maps (Figure 3B) and raw and deconvoluted mass spectra(Figure 4A and Figure 4B) show numerous proteoforms andmuch higher complexity of the sample.
Analysis of the deconvoluted spectra (Figure 3B) corresponding
to each CESI-MS peak (Figure 3A) revealed the presence ofover 202 proteoforms that were predominantly complex, hybridstructures. These data also indicate that the separation andmigration order of PSA molecular species appears to be basedon the hydrodynamic volume and charge of the proteoforms.
In this experiment, we confidently identified 36 glycoforms byhigh-resolution, accurate-mass HRAM MS1 spectra.

CESI-MS under native conditions
Separation of PSA under native conditions yielded results similar
to that of reduced PSA, suggesting that this separation is basedon its hydrodynamic volume and charge distribution. CESI
separation was performed using 40 mM ammonium acetate(pH 7.5) as the background electrolyte, resulting in 5 mainpeaks (Figure 4A). The corresponding ion density map again
illustrated the complexity of PSA (Figure 4B). In this experiment,
the amount of PSA injected was 2.9 ng, representing only
1.14% of the total capillary volume.

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Biomarkers and omics
Analysis of the deconvoluted spectra (Figure 5B) for each peak

6 unique species, although we expected to see many morerevealed the presence of over 236 proteoforms, with the main

glycoforms due to the much greater amount of sample injected.

sialylated biantennary isoform eluting in peak 2. The overall

Since the infusion experiment was performed under nativeseparation profiles under native and denaturing conditions areconditions in which the protein is still folded, it may not bevery similar, strongly suggesting that this separation is based

exposed to enough protons to allow for adequate ionization.

on the hydrodynamic volume and charge distribution of theIn the CESI-MS separation, however, even though less material

protein isotope envelope. Under native conditions, we confidently

was introduced into the capillary, the separation allowed for

identified 38 glycoforms by HRAM MS1 spectra.

better ionization in the native state leading to a greater number

From the 56 identified main glycoforms, 19 species werecommon to the 3 approaches. By CESI-MS under denaturing
and native conditions, we identified 8 and 12 unique glycoforms,
respectively. In the infusion experiment, we detected onlyof unique glycoforms detected. Additionally, no sample
preparation was required other than sample dilution.

Infusion experiments
Using a direct infusion strategy, we characterized the PSA
sample without (Figure 6A) and with (Figure 6B) all ionfragmentation (AIF) using the Exactive Plus EMR system.
Fragment ions, mostly from N and C-termini, generated by AIF
enabled PSA identification (Figure 6C). We achieved sequence
coverage of only 10% because analysis was performed undernative conditions, and PSA tertiary structure was likelyconserved as it contains 5 disulfide bond interactions. This
experiment allowed for the detection and quantification of
only 127 proteoforms by MS1.

Figure 4. (A) Base ion electropherogram of PSA under native conditions,
showing 5 distinct peaks. (B) Density ion map of a separationof PSA under native conditions highlighting the CESI migration
patterns of proteoforms.

Figure 6. PSA nanoLC infusion analysis under native conditions.
(A) MS1 spectrum. (B) AIF spectrum of PSA. (C) PSA sequence,identified b-ions (red) and y-ions (blue) are indicated.
Figure 5. Mass spectra of PSA peaks under native conditions (panel A)
and corresponding deconvoluted spectra (panel B).

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Conclusions

References• CESI is a powerful separation and ionization technique forMS analysis of intact glycoproteins under both native anddenaturing conditions.

1. Leymarie, N. et al. Mol Cell Proteomics. 2013 Oct, 12(10),2935–2951.

• The neutral coated capillary used in this work allowed forthe separation of PSA proteoforms based on their structural
heterogeneity.

2. Viner, R. et al. From Qualitative to Quantitative: The Evolutionof Glycoproteomics. HUPO 2013, Yokohama, poster 51.

• CESI-MS coupled with HRAM MS under both native anddenaturing conditions offers significant improvement for theidentification of glycoforms compared to infusion by nanoLC.
• In this study, we confidently identified and quantified 56 PSA
glycoforms via intact/top-down MS analysis. We also identified
236 proteoforms vs. 127 identified by nanoLC-MS.
• The sample amount loaded in the CESI-MS experiments
was nearly half of that used in a typical nanoLC infusion
Experiment, and yet twice as many proteoforms weredetected. This demonstrates the value of CESI as afront-end separation/ionization tool for these molecules.
• No special sample preparation was required otherthan dilution.

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