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Characterization of Phospho- and Glycopeptides byCESI-MS: A Powerful Technique for Increasing Sensitivity•Increased sensitivity across 3 logs of dynamic range with a low LOD•Reduction in ion suppression bias

Anthonius Heemskerk1 and Mark Lies2
1Leiden University Medical Center, Leiden, Netherlands
2
SCIEX, Brea, CA, USA

Sensitivity in characterization of complex or biologically

Many biologically relevant molecules like immunoglobulins (IgGs)

significant samples is an essential parameter for an overallcan be classified as glycoproteins because they consist of aunderstanding of their heterogeneity, function, and molecular

protein with covalently attached oligosaccharides. Glycosylation

identity. Techniques including capillary electrophoresis (CE) andis a very important characteristic in IgGs as it helps define

liquid chromatography (LC) either as standalone techniques or

biological activity, clearance, and physicochemical properties.

hyphenated with mass spectrometry (MS) have been applied inBecause glycosylation occurs fairly heterogeneously, thebiopharmaceutical and proteomics research. CE has been shown

dynamic range of glycans making up a population of glycoforms

to provide extremely efficient separations but lacked the ability toassociated with a specific IgG can be quite broad, requiring

easily confirm identity without the use of known standards against

highly sensitive analytical techniques for proper characterization.

which to compare. In this regard, CESI-MS, the integration of CE

This requirement, for example, is necessary in cases wherewith electrospray ionization (ESI) into a single dynamic process

an IgG exists at low titer, such as when dealing with antigen-within the same capillary, has been used to reach further into thespecific species.

realm of sensitivity (Busnel et al., 2010). Whereas conventional
CE-MS technology introduces samples into the MS using flow
rates in the hundreds of nanoliters (nl) per minute range, CESIMS can perform this task at ultra-low flow rates of <30 nl/min.
This ultra-low flow rate enables an overall increase in sensitivity
due to the improved presentation of poorly ionizing molecularspecies like glycoproteins and phosphopeptides, effectivelyincreasing ionization efficiency and minimizing ionization
bias (Moini, 2007; Schmidt et al., 2003)). In order to further

Using a transient isotachophoresis (t-ITP) method to enhanceassay sensitivity, digested IgG samples were separated andanalyzed by CESI-MS. The t-ITP-CZE separation provides a
stacking effect prior to separation enabling greater sensitivity
in the analysis due to concentration of material. Interestingly,
the t-ITP-CZE system provided a separation on the basis of theattached glycan (Fig. 1). In contrast, glycopeptides from thedifferent IgG subclasses were found to co-migrate.

investigate this technique, the potential benefits of CESI-MS for
the analysis of biologically significant glycopeptides and also
phosphoproteomics have been studied.

p1

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40Figure 1. (A) Base peak electropherogram of a tryptic digest of total IgG purified from human serum; (B) extracted ion traces of thehigh concentration IgG1 glycopeptide species including assignment; (C) extracted ion traces of the low concentration IgG1 glycopeptide
species including assignment. Blue square, N-acetylglucosamine; red triangle, fucose; green circle, mannose; yellow circle, galactose;
Core-fucosylated
speciesNon-core-fucosylated species
purple diamond, N-acetylneuraminic
acid.

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Applying
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analyzed by CESI-MS and showed variability in core fucosylationand galactosylation (Fig 2). These findings were consistentpathogenic IgG1 alloantibodies produced by pregnant womenwith previously performed nano-RPLC-MS studies which

IgGs in 2these samples demonstrate low core fucosylation,galactosylation (Wuhrer et al, 2008). Remarkably, CESI-MS was
1017able to perform13
these analyses across163 logs of dynamic range
1815
14
9

against 3platelet antigens of the fetus were analyzed. Thea characteristic feature of potential pathogenic importance1

as it promotes platelet destruction and is known to enhance
0ADCC (Shieldset al, 2002). Trypsin digested samples were30321211 heterogeneity
foundin expression of core fucosylation andwith a lower LOD even following a 20-fold sample dilution.

3436Core-fucosylated species
x105
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Non-core-fucosylated species

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3436381540 Time [min]Figure 2. IgG Fc glycosylation profiles of HPA1a alloantibodies purified from sera of three pregnant women. Extracted ion traces are
given of the major core-fucosylated (left) and non-core-fucosylated (right) glycopeptide species. (A) The first HPA1a glycosylation profile
is high in fucosylation, galactosylation, and sialylation, (B) the second shows low fucosylation with high galactosylation and sialylation,and (C) the third shows low galactosylation and sialylation with high fucosylation. Blue square, N-acetylglucosamine; red triangle, fucose;green circle, mannose; yellow circle, galactose; purple diamond, N-acetylneuraminic acid.

p3Protein phosphorylation has long been associated with cellular

MS infusion of a mixture of five synthetic peptides containing

function and cell signaling networks. Because phosphorylation

an increasing number of phosphate groups – 0, 1, 2, 3, or 4

is generally transient in nature, phosphorylated proteins are rare

resulted in more equimolar detection at lower flow rates. In

relative to non-phosphorylated proteins, making analysis difficult

order to define the role of flow rate in MS ionization bias ofwhen interrogating heterogeneous protein populations. Study

these phosphopeptides, the rate of hydrodynamic infusion wasof phosphorylated protein variants by MS can be difficult due tovaried from 4.5 to 500nL/min. This range was chosen keeping

co-elution or co-migration through a column or gradient whichmind that flow rates below 20nL/min have been shown to

can lead to ion suppression in ESI-MS analysis. Additionally,decrease ionization bias in relative peak intensities for somethe degree of phosphorylation also impacts overall ionization

peptides resulting in a trend toward an equimolar response,

efficiency which can be adversely affected by the increasing

a phenomenon that would be valuable in phosphoproteome

negative charge due to the addition of multiple phosphate groups.

analysis (Schmidt et al.). The synthetic phosphopeptides used

Therefore it is assumed that proteins/peptides that are morein this study contained 0 to 4 tyrosine residues to accommodate

heavily phosphorylated are poorly ionizing relative to their non-phosphorylation and were designed with C-terminal arginine

phosphorylated counterparts.

residues to emulate tryptic fragments commonly analyzed inIn order to address questions around differential ionization ofphosphopeptides, Heemskerk et al. (2013) applied ultra lowflow CESI-MS to minimize bias. The objective of the work was
to generate a high resolution separation to better discriminate
phosphorylated from non-phosphorylated species in an ultralow flow environment. Initial research demonstrated that CESI-

peptide mapping experiments. CESI-MS achieved significantly
improved sensitivity for detection of the synthetic phosphopeptide
mixture in conditions where equal sample load and concentration
were used. Importantly, a significant reduction in ionization bias
between the 5 synthetic peptides was seen when decreasing flowrate from >100nl/min to ~10nl/min.

Figure 1. Typical mass spectra of the model phosphopeptide mix infusion at flows below 10 nL/min andabove 100 nL/min.
p4

Further experimentation was performed to examine thisThese data serve to illustrate the powerful capability of CESI-MS

phenomenon using a more complex sample. To accommodate

technology to separate complex phosphopeptide species from

this, a bovine milk digest was analyzed using transient

one another.

isotachophoresis (t-ITP). Bovine milk is a complex biological

Taken together, the data presented in this Technical Note using

sample with a large proportion of the protein content

the CESI 8000 High Sensitivity ESI Module describes thecorresponding to αS1, αS2, β and κ caseins, on which there

capability of an ultra-low flow environment to attain significant

exist abundant phosphorylations. t-ITP-ESI-MS allowed

increases in sensitivity while decreasing ionization bias amongfor 250 nl of sample (~37% of the capillary) to be injected,

analytes. As specifically highlighted, both phosphopeptides andresulting in protein stacking and therefore higher concentration

glycoproteins, which generally are challenging to analyze using

for introduction into the MS. As higher concentration protein

mass spectrometry, can be efficiently ionized and detected withsamples have a tendency to precipitate or interact with theminimal bias in CESI-MS. It should also be noted that no sample

capillary surface, CESI-MS was performed using a separation

pre-concentration was performed to achieve this sensitivity

capillary with a chemically neutral surface. While the neutral

increase and ionization improvement, thus simplifying thesurface helps minimize protein-wall interactions, it also serves

overall workflow from sample to data.

to virtually eliminate electroosmotic flow (EOF), helping extend
the separation window and therefore maximize the resolving
capability. Loading 5ng of the bovine milk digest, CESI-MS
was capable of identifying 13 phosphopeptides, including 2 multiphosphorylated peptides, in comparison to only 5 phosphorylated
peptides identified using nano-LC-MS (Heemskerk et al, 2012).
In fact, even a 10x increase in sample load in LC-MS could notequal the sensitivity of CESI-MS.

Analysis characteristics

Number ofphosphopeptides

1P2PUniquephosphopeptides

5 ng milk digest tITP-MS

1210255 ng milk digest nano-LC-MS

550150 ng milk digest nano-LC-MS

9903

Table 1. A comparison of the detected phosphopeptides from a bovine milk digest.
(Table adapted from Heemskerk et al, 2012)p5

References
Busnel, J.-M.; Schoenmaker, B.; Ramautar, R.; CarrascoPancorbo, A.; Ratnayake, C.; Feitelson, J. S.; Chapman,J. D.; Deelder, A. M.; Mayboroda, O. A. Analytical Chemistry
2010, 82 (22), p9476-9483.
Heemskerk AM; Wuhrer M; Busnel J-M; Koeleman C; Selman M;
Vidarsson G; Kapur R; Schoenmaker B; Derks R; Deelder AM;
Mayboroda O. (2013) Electrophoresis, 34, 3, p383–387.
Heemskerk AM; Busnel J-M; Schoenmaker B; Derks R;
Klychnikov O; Hensbergen P; Deelder A; Mayboroda O.
Analytical Chemistry (2012), 84 (10), p 4552–4559.
Moini, M., Analytical Chemistry (2007), 79 (11), p4241-4246.
Schmidt, A.; Karas, M.; Dulcks, T. Journal of the American
Society for Mass Spectrometry (2003), 14 (5), p492-500.
Shields, R. L., Lai, J., Keck, R., O’Connell, L. Y., Hong, K.,Meng, Y. G., Weikert, S. H. A., Presta, L. G., J. Biol. Chem.
(2002), 277, p26733–26740.
Wuhrer, M., Porcelijn, L., Kapur, R., Koeleman, C. A. M., Deelder,A. M., de Haas, M., Vidarsson, G., J. Proteome Res. (2008), 8,
p450–456.

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