Technical Note pubs.acs.org/jpr
Differential Isotope Labeling of Glycopeptides for Accurate Determination of Differences in Site-Specific Glycosylation Martin Pabst,†,# Iva Benešová,†,‡,# Stephan R. Fagerer,† Mathias Jacobsen,† Klaus Eyer,† Gregor Schmidt,§ Robert Steinhoff,† Jasmin Krismer,† Fabian Wahl,∥ Jan Preisler,‡,⊥ and Renato Zenobi*,† †
Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland Department of Chemistry, Masaryk University, 625 00 Brno, Czech Republic § Department of Biosystems Science and Engineering (D-BSSE), ETH Zurich, 4058 Basel, Switzerland ∥ Sigma-Aldrich Chemie GmbH, 9470 Buchs, Switzerland ⊥ Central European Institute of Technology (CEITEC), Masaryk University, 625 00 Brno, Czech Republic ‡
S Supporting Information *
ABSTRACT: We introduce a stable isotope labeling approach for glycopeptides that allows a specific glycosylation site in a protein to be quantitatively evaluated using mass spectrometry. Succinic anhydride is used to specifically label primary amino groups of the peptide portion of the glycopeptides. The heavy form (D413C4) provides an 8 Da mass increment over the light natural form (H412C4), allowing simultaneous analysis and direct comparison of two glycopeptide profiles in a single MS scan. We have optimized a protocol for an in-solution trypsin digestion, a one-pot labeling procedure, and a post-labeling solid-phase extraction to obtain purified and labeled glycopeptides. We provide the first demonstration of this approach by comparing IgG1 Fc glycopeptides from polyclonal IgG samples with respect to their galactosylation and sialylation patterns using MALDI MS and LC−ESI−MS.
KEYWORDS: glycopeptides, stable isotope labeling, IgG, MALDI MS, LC−ESI−MS
■
spectrum.8,9 For quantitative and reproducible MS analyses, homogeneous crystallization of sample spots or a robust spray are crucial in MALDI MS (matrix-assisted laser desorption/ ionization mass spectrometry) or ESI-MS (electrospray ionization mass spectrometry), respectively. Therefore, even samples of the same origin that have been prepared and analyzed in parallel may show some artificial differences in their observed profiles. The use of a stable isotope label allows the pooling of at least two samples at an early stage of the sample workup. Bias introduced by sample preparation and cleanup, MALDI spot crystallization, or ionization processes will affect analytes from both samples to the same degree. Also, absolute quantification of glycoforms can be realized by employing a stable isotope labeled internal standard, which is characterized by an orthogonal approach such as glycan release and fluorescence labeling. This would allow the glycoform quantities for an unknown sample to be accurately determined based on glycopeptides by eliminating factors such as in-source fragmentation or ionization processes. Differential isotope labeling is already well-established in proteomics10−12 and has also been successfully demonstrated
INTRODUCTION In order to monitor differences or alterations at a specific glycosylation site of a protein, a mass spectrometric approach focusing on glycopeptides is necessary.1−6 A released glycan pool, on the other hand, delivers only an overall spectrum of all glycosites present, which might also be influenced by glycoprotein contaminants.2,7 The quantitative investigation of a specific glycosylation site in a protein is required when monitoring alterations in the glycoform distribution during disease or a developmental stage. In addition, it might be of value to determine the absolute abundances of the individual glycoforms present at a distinct glycosite responsible for protein−protein or protein−cell interactions. In quantitative investigations, sample preparation and analysis have to be chosen carefully and should ideally be highly reproducible and nondestructive. Purification and analyte enrichment steps may enhance selectivity and sensitivity, but there is also risk in favoring or suppressing certain glycoforms and in this way introducing a bias. Furthermore, obtaining absolute values for individual glycoforms is particularly difficult using a pure mass spectrometric approach. In addition to sample preparation, ionization processes, in-source or postsource decay, and instrumentdependent parameters can also lead to a more or less distorted © XXXX American Chemical Society
Received: April 22, 2015
A
DOI: 10.1021/acs.jproteome.5b00899 J. Proteome Res. XXXX, XXX, XXX−XXX
Technical Note
Journal of Proteome Research recently for released glycans.13−15 Here, we describe for the first time the use of differential isotope labeling for glycopeptides. Succinic anhydride (SA, natural isotope form and d4, with a mass difference of 4 Da) was first introduced by Zhang et al.16 for labeling covalently captured glycopeptides, where the glycan portion was then released prior to analysis to determine differences in the degree of glycosylation using LC−ESI−MS/ MS (liquid chromatography coupled to electrospray ionization (tandem) mass spectrometry). IgG Fc glycosylation can be analyzed by mass spectrometry using ESI−MS or MALDI MS, with or without prior separation;5,6,17−19 as well as after glycan release and analysis of the native glycans by MALDI MS, HILIC-MS (hydrophilic interaction liquid chromatography coupled to MS), and porous graphitized carbon chromatography coupled to MS or after fluorescence labeling and HILIC/normal-phase chromatography with fluorescence detection.8,20−25 Several diseases have been found to be linked to alterations in IgG Fc glycosylation, often showing a reduced degree of galactosylation or sialylation.26−28 Recent findings show that the degree of sialylation is significant and could even determine whether IgG acts in either a pro- or anti-inflammatory manner.29−31 Fc glycosylation was, therefore, considered to be a kind of carbohydrate switch, where only subtle differences dictate whether the substance acts as pathogenic or antigenic.29 However, polyclonal IgGs are known to present approximately 20% Fab glycosylation.6,32,33 Fc and Fab glycosylation can differ significantly and can also be influenced to a different extent;34 therefore, a released glycan pool does not reflect the true picture of the IgG Fc glycosylation of a particular subclass. Here, we give the first demonstration of a differential isotope labeling approach for glycopeptides by evaluating IgG1 Fc glycosylation of several polyclonal IgG samples (intravenous immunoglobulin, IVIg) with respect to galactosylation and sialylation. We further describe a one-pot labeling protocol for tryptic peptides and a post-labeling solid-phase extraction using ZIC-HILIC (zwitterionic hydrophilic interaction liquid chromatography) solid-phase extraction cartridges. Analysis of the labeled glycopeptides is then demonstrated using MALDI MS and nano-LC−ESI−MS.
■
added to the reaction mixture in order to carbamidomethylate the sulfhydryl groups. Proteins were then precipitated with 800 μL of acetone (20 min, −20 °C) and centrifuged at maximum speed for 10 min. The pellet was washed two times with icecold acetone and dried in a SpeedVac concentrator. Tryptic digestion was carried out in 500 μL of 25 mM NH4HCO3 with 2 U each of trypsin (Sigma, cat. no. T6567) at 37 °C overnight. The digest was aliquoted into separate tubes, each containing 50 μg of the digested protein, and dried under vacuum. The residual NH4HCO3 was removed in two cycles of washing using 50 μL of 50% acetonitrile, followed by vacuum drying in a SpeedVac concentrator. Neuraminidase Treatment
Aliquots from the IVIg Privigen samples were artificially desialylated using a 2-3/6-neuraminidase obtained from Sigma-Aldrich. For this, the tryptic peptides were dissolved in 100 μL of reaction buffer (1×) with 2 μL (1 mU) of neuraminidase solution and incubated at 37 °C overnight. Neuraminidase was finally inactivated by briefly heating the sample at 96 °C. In parallel, control samples were incubated in the reaction buffer only in order to investigate nonenzymatic degradation during incubation. Desialylated samples were then mixed with the control (untreated) samples to obtain a set of samples differing in their degree of sialylation (0−50% sialylation). All prepared samples were vacuum-dried in a SpeedVac concentrator. The reaction buffer was removed with a further washing step using 50 μL of 50% acetonitrile followed by a second vacuum drying. Labeling Protocol
Glycopeptides in the tryptic digest were labeled with succinic anhydride (SA) using a modified protocol of that originally described by Zhang et al.16 Vacuum-dried tryptic digests (typically, 50 μg of total protein) were resuspended in DMF/ pyridine/H2O (50:10:40 [v/v/v]; 18 μL) and mixed with succinic anhydride solution (2 μL) to a final concentration of 2 mg/mL anhydride. The samples were incubated at room temperature for 1 h before the reaction was quenched with 20 μL of 2% acetonitrile in H2O with 0.2% formic acid. Individual samples were then mixed in equal amounts according to the starting material. The same procedure was used for the light form SA (SA0) as well as for the heavy form SA-d413C4 (SA8). The light form results from the addition of 100.01 Da, and the heavy form, from the addition of 108.05 Da to each primary amino group of a peptide.
MATERIALS AND METHODS
Stable Isotope Labeling Using Succinic Anhydride
Succinic anhydride (SA0, natural isotopic composition) was obtained from Sigma-Aldrich Chemie GmbH (Buchs, Switzerland). D413C4 (SA8) labeled succinic anhydride was prepared from 13C4, 2.2.3.3-d4 succinic acid by treatment with acetic anhydride as described in Gajewski and Salazar.35 13C4, 2.2.3.3d4 succinic acid was purchased from EURISO-TOP (Saint-Au bin Cedex, France).
Glycopeptide Purification
The labeled glycopeptides were further purified and enriched using ZIC-HILIC solid-phase extraction (SeQuant, Sweden, 1 mL bed size) based on the protocol described in Parker et al.37 SPE (solid-phase extraction) columns were conditioned using 1 mL of 80% acetonitrile in H2O with 0.1% trifluoroacetic acid (equilibration solution), then with 1 mL water, and finally once more with 1 mL of the equilibration solution. A mixture of SA0 and SA8 labeled digests was diluted to 800 μL with the equilibration solution, loaded on the SPE column, and washed with 750 μL of the equilibration solution. Glycopeptides were finally eluted with 300 μL of water, SpeedVac dried, and redissolved in water to a final concentration of 1 μg (total protein)/μL.
In-Solution Trypsin Digestion
All IgG samples (250 μg each) were desalted using 30 kDa cutoff spin columns (Amicon Ultra 0.5 mL centrifugal filters, Merck Millipore), as recommended by the manufacturer, prior to proteolytic digestion. For this, samples were diluted to a final volume of 500 μL, centrifuged at maximum speed for 15 min, and washed once with 250 μL of water. The protein was recovered by a reverse spin with 25 μL of 0.1 M NH4HCO3. The desalted proteins were digested as described in Grass et al.36 Briefly, polyclonal IgG was reduced with a solution of 100 μL of 15 mM dithiothreitol in 0.1 M NH4HCO3 at 56 °C for 45 min. 100 μL of an iodoacetamide solution (110 mM) was
MALDI MS
MALDI MS analysis was carried out using an AB 5800 mass spectrometer in reflectron negative and positive modes. Labeled B
DOI: 10.1021/acs.jproteome.5b00899 J. Proteome Res. XXXX, XXX, XXX−XXX
Technical Note
Journal of Proteome Research
Figure 1. Succinic anhydride reacts with the primary amino group of the peptide to form a stable amide bond. The light version increased the mass by 100.01 Da (SA0), whereas the heavy form shifts the mass by 108.05 Da (SA8). The final 8.04 Da mass difference between heavy and light forms allows a combined measurement of two samples without interference from natural isotope or adduct peaks in the MS scan. This allows a direct comparison of two samples to be made with regard to their glycoform distributioin at a distinct glycosylation site in the protein or enables the further employment of an orthogonal validated internal (glycopeptide) standard.
glycopeptides were spotted on a stainless steel target (0.5 μL) and air-dried. Subsequently, the sample spot was covered with 0.5 μL of 4-chloro-α-cyanocinnamic acid (ClCCA; 5 mg/mL and dissolved in a solution of 70% acetonitrile in H2O) matrix. The sample was dried under ambient conditions and analyzed using a laser energy of 4500 (arbitrary units). The crude glycopeptide fractions were analyzed using 2,5-dihydroxybenzoic acid (DHB; 10 mg/mL in 50% acetonitrile with 1% phosphoric acid), where phosphoric acid was found to be beneficial as an additive in a similar way as that described recently for unlabeled glycopeptides and phosphopeptides.38−40 A mass spectrum was obtained by averaging over 100 spot positions with 20 shots per each subspectrum. Focus mass was set to 2500 Da, and delayed extraction was set to 500 ns. Mass spectra were further analyzed using the DataExplorer software from AB Sciex and by using mMass (www.mmass.org).41
from polyclonal IgG (Privigen) were mixed with uniform reagent compositions and volumes. For amounts between 5 and 100 μg of total glycoprotein digest, samples were labeled with very similar efficiency and reproducibility (>90%, based on MALDI MS; Figures S1 and 2). Byproducts due to unintended anhydride reaction were not observed. As expected, each observed glycopeptide series (only the most abundant peaks from the IgG1 and IgG2 were annotated) resulted in a mass increase of 100.01 Da (addition of the light form, SA0). The ionization efficiency in MALDI MS and the retention behavior from reverse-phase chromatography of the labeled glycopeptides are described in the Supporting Information (effect of the SA label on ionization efficiency in MALDI; reverse-phase analysis and deuterium retention effect; Figure S3). Polyclonal human IgG is a rather complex sample that can be classified into four subclasses (IgG1−4), which all have a conserved N-glycosylation site on the heavy chain constant region.32,33 The tryptic Fc glycosite peptide from IgG1 heavy chain (P01857) has the conserved sequence EEQYNSTYR, the IgG2 heavy chain (P01859) has the conserved sequence EEQFNSTFR, and IgG3 (P01860) has the sequence EEQY*NSTFR, where Yuan et al. have shown that Y* can also be replaced by F due to a polymorphic gene variant. This is varied in its frequency in a population and is also expressed to differing extents in individuals and therefore shows either both forms or just the wild type.42 IgG4 (P01861) tryptic Fc glycopeptide shows the conserved sequence EEQFNSTYR. Labeling quality and progress were monitored directly by MALDI MS analysis of small aliquots of the reaction mixture. For this, DHB, which is more tolerant toward the presence of salts, proved to be a useful MALDI matrix. Labeled and unlabeled samples (SA8 and SA0) were pooled in order to perform a combined sample cleanup. To do so, the quenched sample solutions were combined and extracted using a ZICHILIC solid-phase extraction cartridge. ClCCA was found to be the most suitable MALDI matrix for the purified glycopeptides, providing the highest S/N ratio. In the first evaluation of the method, a single sample was split once before tryptic digestion and once just before labeling. In both cases, the samples were
Nano-LC−ESI−MS
Chromatographic separation was carried out using an Eksigent ekspert nano-LC 415 with a reverse-phase column (0.075 × 150 mm, Eksigent RPC18-CL-120). Solvent A comprised a solution of 0.1% unbuffered formic acid and 2% ACN in H2O, and solvent B, a solution of 95% acetonitrile in H2O. An aliquot of 5 μL of protein mix (prepared as described above) containing close to 1 μg of glycopeptide mixture was injected into the nano-LC system and separated using a gradient from 5 to 35% B during 15 min at a flow rate of 400 nL/min. The outlet of the nano-LC column was connected to an atmospheric pressure nanosprayer interface of a Synapt G2S mass spectrometer (Waters, Milford, USA). The samples were analyzed in sensitivity mode at medium resolution over a mass range of 500−2000 Da at a 2 Hz sampling rate. Glycopeptide mass peak intensities and ratios were further evaluated using MassLynx 4.2 software (Waters) and Microsoft Excel.
■
RESULTS AND DISCUSSION
Stable Isotope Labeling Approach
In order to test the suitability of the labeling approach as demonstrated in Figure 1, different amounts of tryptic digests C
DOI: 10.1021/acs.jproteome.5b00899 J. Proteome Res. XXXX, XXX, XXX−XXX
Technical Note
Journal of Proteome Research
Figure 2. IgG glycopeptides prior to (spectrum A, G1F(IgG1) underivatized = 2796) and (spectrum B) after labeling (G1F(IgG1) SA0 = 2896, SA8 = 2904). The 8.04 Da mass difference between heavy and light forms was sufficient to measure the IgG Fc glycopeptides without interference from natural isotope or adduct peaks.
pooled again after differential labeling and analyzed by MALDI MS. No obvious differences in the overall glycoprofile were noticed, neither for the pair split before tryptic digestion nor for the pair split directly before labeling. This demonstrates that bias, if any, introduced by the proteolytic digestion step of the samples was minimal and furthermore that the labeling protocol itself does not lead to bias in the glycopeptide profile (Figure 2). Comparison of Different Polyclonal IgG Samples
In addition, we investigated differences between the Fc glycosylation profiles of different Privigen batches and those obtained from a chemical supplier. These are produced by pooling a large population of donor samples; therefore, the differences were expected to be minimal. We focused our investigation primarily on the degree of galactosylation and sialylation. The differences in overall galactosylation could be observed with MALDI MS as well as by nano-LC−ESI−MS. The slight differences in sialylation were noticed only with the nano-LC−ESI−MS approach due to in-source decay of the sialylated species in MALDI (Figures 3 and S4). Although the direct MALDI MS approach is much faster, it has clear limitations due to the potential in-source fragmentation of labile residues such as sialic acids and due to potential superposition of mass peaks originating from different glycopeptide series. We additionally generated a set of IgG samples that differed in their overall degree of sialylation. For this, a portion of the tryptic digest was desialylated using neuraminidase prior to the stable isotope labeling and mixed with an untreated sample to obtain one sample with 50% reduced sialylation, one with 25% reduced sialylation, and a further one with 10% reduced sialylation. Another sample containing an inactivated neuraminidase was used as a control (for further information, see Figures S5−S7). Considering that the sialylated species are rather low-abundance, a decrease in 10% of a minor form
Figure 3. Differences in the degrees of galactosylation and sialylation present on the Fc regions of human polyclonal IgG1 analyzed via nano-LC−ESI−MS. For each glycoform, the signal intensity ratio with its internal SA8 standard was calculated and then normalized to the intensity of the G0F glycoform. Samples: left bar represents Privigen batch 1 (used also as SA8 internal standard for the other two samples), the middle bar shows the levels of another Privigen batch, and the right bar shows a human polyclonal IgG sample obtained from a chemical supplier. In addition to slight changes in the degree of galactosylation, we observed an increase in sialylation for the second Privigen batch and a slightly lower degree of sialylation for the human polyclonal IgG sample obtained from a chemical supplier.
would be challenging to differentiate even after glycan release, fluorescence labeling, and HPLC with fluorescence detection analysis. Furthermore, the sialylated species were hardly detected at all using the direct MALDI MS approach, presumably due to insource decay, as mentioned above. For the instrumental setup used here, this was true for both positive and negative ionization modes. However, this might be improved by employing a different MALDI or matrix system. Nevertheless, D
DOI: 10.1021/acs.jproteome.5b00899 J. Proteome Res. XXXX, XXX, XXX−XXX
Journal of Proteome Research
■
the decrease in sialylation could be indirectly noticed by an increase in the free galactosylated structures G2F (AAF6) and G1F (AGnF6/GnAF6), as shown in Figure S5. Using LC−ESI−MS, on the other hand, allowed us to monitor the expected decrease in sialylation based on the G2SF (NaAF6/ANaF6) mass signals even for the sample showing a 10% change (Figures 4 and S6).
Technical Note
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.5b00899. Figure S1: Monitoring the labeling reaction and effect of the SA-label on ionization; Figure S2: Precision; Figure S3: Deuterium retention effect; Figure S4: Degree of galactosylation by MALDI MS; Figure S5: Degree of sialylation by MALDI MS; Figure S6: ESI-MS spectra for sialylated species; Figure S7: Neuraminidase-treated sample (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: +41446324376. Author Contributions #
M.P. and I.B. contributed equally to this work.
Notes
Figure 4. Nano-LC−ESI−MS analysis of the test set with different degrees of IgG1 Fc sialylation (the left bar shows the untreated control sample, the middle bar shows a sample with 10% decreased sialylation, and the right bar represents a sample with 50% decreased sialylation). G1F (AGnF/GnAF) and G2F (AAF) were found to be increased, where the degree of sialylation decreased according to the treatment of neuraminidase. For each glycoform, the signal intensity ratio with its internal SA8 standard was calculated and then normalized to the intensity of the G0F (GnGn) intensity ratio. MALDI MS data for the same set of samples is presented in Figure S3.
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work was supported by the Swiss KTI (Kommission für Technologie und Innovation; grant no. 13123.1 PFNM-NM) and by the programs “Employment of Newly Graduated Doctors of Science for Scientific Excellence” (CZ.1.07/2.3.00/ 37130.0009) and CEITEC (CZ.1.05/1.1.00/02.0068). We would like to thank Tobias Schibli from AB Sciex for loaning the Eksigent ekspert nano-LC 415 and the AB 5800 MALDI MS instruments to us.
■
■
SUMMARY We introduce and describe succinic anhydride as a stable isotope label for glycopeptides to quantitatively evaluate glycan profiles at a specific glycosylation site of a protein. Stable isotope labeling allows at least two samples at an early stage to be pooled; therefore, bias introduced by sample preparation, MALDI spot crystallization, or ionization processes affects both samples to the same degree. This approach thus allows minimal differences/alterations in the glycan distribution of a specific site to be monitored with high confidence. Succinic anhydride reacts via amino groups of peptides without affecting the glycan portion itself. In order to obtain a suitable change in the mass increment between a labeled pair, we chose succinic anhydride in its natural isotope form and as d413C4 (delta of 8 Da). The pooled and labeled glycopeptides were analyzed by MALDI MS and nano-LC−ESI−MS. We showed the first applications of this approach by analyzing IgG1 Fc glycosylation of various polyclonal IgG samples and additionally demonstrated its capability to monitor small changes in the degree of galactosylation and sialylation. The stable isotope labeling approach presented here could also be advantageous for proteins with a larger number of glycosylation sites and for samples with a higher degree of complexity in their glycosylation profiles. Moreover, a set of stable isotope labeled standards could be validated with an orthogonal method like glycan release and fluorescence labeling. This standard could then allow selected glycoforms or glycoform ratios at a specific glycosylation site within a protein to be quantitatively determined from a low amount of sample not achievable with common methods.
REFERENCES
(1) Kolarich, D.; Jensen, P. H.; Altmann, F.; Packer, N. H. Determination of site-specific glycan heterogeneity on glycoproteins. Nat. Protoc. 2012, 7, 1285−98. (2) Pabst, M.; Chang, M.; Stadlmann, J.; Altmann, F. Glycan profiles of the 27 N-glycosylation sites of the HIV envelope protein CN54gp140. Biol. Chem. 2012, 393, 719−30. (3) Stadlmann, J.; Pabst, M.; Kolarich, D.; Kunert, R.; Altmann, F. Analysis of immunoglobulin glycosylation by LC−ESI−MS of glycopeptides and oligosaccharides. Proteomics 2008, 8, 2858−71. (4) Marino, K.; Bones, J.; Kattla, J. J.; Rudd, P. M. A systematic approach to protein glycosylation analysis: a path through the maze. Nat. Chem. Biol. 2010, 6, 713−23. (5) Selman, M. H.; Derks, R. J.; Bondt, A.; Palmblad, M.; Schoenmaker, B.; Koeleman, C. A.; van de Geijn, F. E.; Dolhain, R. J.; Deelder, A. M.; Wuhrer, M. Fc specific IgG glycosylation profiling by robust nano-reverse phase HPLC-MS using a sheath-flow ESI sprayer interface. J. Proteomics 2012, 75, 1318−29. (6) Wuhrer, M.; Stam, J. C.; van de Geijn, F. E.; Koeleman, C. A.; Verrips, C. T.; Dolhain, R. J.; Hokke, C. H.; Deelder, A. M. Glycosylation profiling of immunoglobulin G (IgG) subclasses from human serum. Proteomics 2007, 7, 4070−81. (7) Pucic, M.; Knezevic, A.; Vidic, J.; Adamczyk, B.; Novokmet, M.; Polasek, O.; Gornik, O.; Supraha-Goreta, S.; Wormald, M. R.; Redzic, I.; Campbell, H.; Wright, A.; Hastie, N. D.; Wilson, J. F.; Rudan, I.; Wuhrer, M.; Rudd, P. M.; Josic, D.; Lauc, G. High throughput isolation and glycosylation analysis of IgG-variability and heritability of the IgG glycome in three isolated human populations. Mol. Cell. Proteomics 2011, 10, M111.010090. (8) Leymarie, N.; Griffin, P. J.; Jonscher, K.; Kolarich, D.; Orlando, R.; McComb, M.; Zaia, J.; Aguilan, J.; Alley, W. R.; Altmann, F.; Ball, L. E.; Basumallick, L.; Bazemore-Walker, C. R.; Behnken, H.; Blank,
E
DOI: 10.1021/acs.jproteome.5b00899 J. Proteome Res. XXXX, XXX, XXX−XXX
Technical Note
Journal of Proteome Research M. A.; Brown, K. J.; Bunz, S. C.; Cairo, C. W.; Cipollo, J. F.; Daneshfar, R.; Desaire, H.; Drake, R. R.; Go, E. P.; Goldman, R.; Gruber, C.; Halim, A.; Hathout, Y.; Hensbergen, P. J.; Horn, D. M.; Hurum, D.; Jabs, W.; Larson, G.; Ly, M.; Mann, B. F.; Marx, K.; Mechref, Y.; Meyer, B.; Moginger, U.; Neususs, C.; Nilsson, J.; Novotny, M. V.; Nyalwidhe, J. O.; Packer, N. H.; Pompach, P.; Reiz, B.; Resemann, A.; Rohrer, J. S.; Ruthenbeck, A.; Sanda, M.; Schulz, J. M.; Schweiger-Hufnagel, U.; Sihlbom, C.; Song, E.; Staples, G. O.; Suckau, D.; Tang, H.; Thaysen-Andersen, M.; Viner, R. I.; An, Y.; Valmu, L.; Wada, Y.; Watson, M.; Windwarder, M.; Whittal, R.; Wuhrer, M.; Zhu, Y.; Zou, C. Interlaboratory study on differential analysis of protein glycosylation by mass spectrometry: the ABRF glycoprotein research multi-institutional study 2012. Mol. Cell. Proteomics 2013, 12, 2935−51. (9) Stavenhagen, K.; Hinneburg, H.; Thaysen-Andersen, M.; Hartmann, L.; Silva, D. V.; Fuchser, J.; Kaspar, S.; Rapp, E.; Seeberger, P. H.; Kolarich, D. Quantitative mapping of glycoprotein micro-heterogeneity and macro-heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides. J. Mass Spectrom. 2013, 48, 627−39. (10) Tao, W. A.; Aebersold, R. Advances in quantitative proteomics via stable isotope tagging and mass spectrometry. Curr. Opin. Biotechnol. 2003, 14, 110−8. (11) Bantscheff, M.; Schirle, M.; Sweetman, G.; Rick, J.; Kuster, B. Quantitative mass spectrometry in proteomics: a critical review. Anal. Bioanal. Chem. 2007, 389, 1017−31. (12) Julka, S.; Regnier, F. Quantification in proteomics through stable isotope coding: a review. J. Proteome Res. 2004, 3, 350−63. (13) Bowman, M. J.; Zaia, J. Tags for the stable isotopic labeling of carbohydrates and quantitative analysis by mass spectrometry. Anal. Chem. 2007, 79, 5777−84. (14) Xia, B.; Feasley, C. L.; Sachdev, G. P.; Smith, D. F.; Cummings, R. D. Glycan reductive isotope labeling for quantitative glycomics. Anal. Biochem. 2009, 387, 162−70. (15) Michael, C.; Rizzi, A. M. Quantitative isomer-specific N-glycan fingerprinting using isotope coded labeling and high performance liquid chromatography-electrospray ionization-mass spectrometry with graphitic carbon stationary phase. J. Chromatogr A 2015, 1383, 88−95. (16) Zhang, H.; Li, X. J.; Martin, D. B.; Aebersold, R. Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat. Biotechnol. 2003, 21, 660−6. (17) Bakovic, M. P.; Selman, M. H.; Hoffmann, M.; Rudan, I.; Campbell, H.; Deelder, A. M.; Lauc, G.; Wuhrer, M. High-throughput IgG Fc N-glycosylation profiling by mass spectrometry of glycopeptides. J. Proteome Res. 2013, 12, 821−31. (18) Zauner, G.; Selman, M. H.; Bondt, A.; Rombouts, Y.; Blank, D.; Deelder, A. M.; Wuhrer, M. Glycoproteomic analysis of antibodies. Mol. Cell. Proteomics 2013, 12, 856−65. (19) Reusch, D.; Haberger, M.; Maier, B.; Maier, M.; Kloseck, R.; Zimmermann, B.; Hook, M.; Szabo, Z.; Tep, S.; Wegstein, J.; Alt, N.; Bulau, P.; Wuhrer, M. Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles–part 1: separation-based methods. MAbs 2015, 7, 167−79. (20) Royle, L.; Campbell, M. P.; Radcliffe, C. M.; White, D. M.; Harvey, D. J.; Abrahams, J. L.; Kim, Y. G.; Henry, G. W.; Shadick, N. A.; Weinblatt, M. E.; Lee, D. M.; Rudd, P. M.; Dwek, R. A. HPLCbased analysis of serum N-glycans on a 96-well plate platform with dedicated database software. Anal. Biochem. 2008, 376, 1−12. (21) Pabst, M.; Altmann, F. Glycan analysis by modern instrumental methods. Proteomics 2011, 11, 631−43. (22) Costello, C. E.; Contado-Miller, J. M.; Cipollo, J. F. A glycomics platform for the analysis of permethylated oligosaccharide alditols. J. Am. Soc. Mass Spectrom. 2007, 18, 1799−812. (23) Harvey, D. J. Proteomic analysis of glycosylation: structural determination of N- and O-linked glycans by mass spectrometry. Expert Rev. Proteomics 2005, 2, 87−101.
(24) Jensen, P. H.; Karlsson, N. G.; Kolarich, D.; Packer, N. H. Structural analysis of N- and O-glycans released from glycoproteins. Nat. Protoc. 2012, 7, 1299−310. (25) Morelle, W.; Faid, V.; Chirat, F.; Michalski, J. C. Analysis of Nand O-linked glycans from glycoproteins using MALDI-TOF mass spectrometry. Methods Mol. Biol. 2009, 534, 3−21. (26) Chen, G.; Wang, Y.; Qiu, L.; Qin, X.; Liu, H.; Wang, X.; Wang, Y.; Song, G.; Li, F.; Guo, Y.; Li, F.; Guo, S.; Li, Z. Human IgG Fcglycosylation profiling reveals associations with age, sex, female sex hormones and thyroid cancer. J. Proteomics 2012, 75, 2824−34. (27) Lux, A.; Nimmerjahn, F. Impact of differential glycosylation on IgG activity. Adv. Exp. Med. Biol. 2011, 780, 113−24. (28) Selman, M. H.; Niks, E. H.; Titulaer, M. J.; Verschuuren, J. J.; Wuhrer, M.; Deelder, A. M. IgG fc N-glycosylation changes in Lambert-Eaton myasthenic syndrome and myasthenia gravis. J. Proteome Res. 2011, 10, 143−52. (29) Collin, M.; Ehlers, M. The carbohydrate switch between pathogenic and immunosuppressive antigen-specific antibodies. Exp Dermatol 2013, 22, 511−4. (30) Kaneko, Y.; Nimmerjahn, F.; Ravetch, J. V. Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation. Science 2006, 313, 670−3. (31) Stadlmann, J.; Pabst, M.; Altmann, F. Analytical and Functional Aspects of Antibody Sialylation. J. Clin. Immunol. 2010, 30, 15−9. (32) Oxelius, V. A. Immunoglobulin G (IgG) subclasses and human disease. Am. J. Med. 1984, 76, 7−18. (33) Stadlmann, J.; Weber, A.; Pabst, M.; Anderle, H.; Kunert, R.; Ehrlich, H. J.; Peter Schwarz, H.; Altmann, F. A close look at human IgG sialylation and subclass distribution after lectin fractionation. Proteomics 2009, 9, 4143−53. (34) Bondt, A.; Rombouts, Y.; Selman, M. H.; Hensbergen, P. J.; Reiding, K. R.; Hazes, J. M.; Dolhain, R. J.; Wuhrer, M. IgG Fab glycosylation analysis using a new mass spectrometric high-throughput profiling method reveals pregnancy-associated changes. Mol. Cell. Proteomics 2014, 13, 3029. (35) Gajewski, J. J.; Salazar, J. D. C. Degenerate thermal rearrangement of 1, 3-dimethylenecyclopentane. Evidence for partially stereospecific biradical formation and closure in a 1, 3 shift. J. Am. Chem. Soc. 1981, 103, 4145−4154. (36) Grass, J.; Pabst, M.; Chang, M.; Wozny, M.; Altmann, F. Analysis of recombinant human follicle-stimulating hormone (FSH) by mass spectrometric approaches. Anal. Bioanal. Chem. 2011, 400, 2427−38. (37) Parker, B. L.; Palmisano, G.; Edwards, A. V.; White, M. Y.; Engholm-Keller, K.; Lee, A.; Scott, N. E.; Kolarich, D.; Hambly, B. D.; Packer, N. H.; Larsen, M. R.; Cordwell, S. J. Quantitative N-linked glycoproteomics of myocardial ischemia and reperfusion injury reveals early remodeling in the extracellular environment. Mol. Cell. Proteomics 2011, 10, M110.006833. (38) Kuster, S. K.; Pabst, M.; Jefimovs, K.; Zenobi, R.; Dittrich, P. S. High-resolution droplet-based fractionation of nano-LC separations onto microarrays for MALDI-MS analysis. Anal. Chem. 2014, 86, 4848−55. (39) Kjellstrom, S.; Jensen, O. N. Phosphoric acid as a matrix additive for MALDI MS analysis of phosphopeptides and phosphoproteins. Anal. Chem. 2004, 76, 5109−17. (40) Pabst, M.; Kuster, S. K.; Wahl, F.; Krismer, J.; Dittrich, P. S.; Zenobi, R. A Microarray-Matrix-assisted Laser Desorption/IonizationMass Spectrometry Approach for Site-specific Protein N-glycosylation Analysis, as Demonstrated for Human Serum Immunoglobulin M (IgM). Mol. Cell. Proteomics 2015, 14, 1645−56. (41) Strohalm, M.; Kavan, D.; Novak, P.; Volny, M.; Havlicek, V. mMass 3: a cross-platform software environment for precise analysis of mass spectrometric data. Anal. Chem. 2010, 82, 4648−51. (42) Yuan, W.; Sanda, M.; Wu, J.; Koomen, J.; Goldman, R. Quantitative analysis of immunoglobulin subclasses and subclass specific glycosylation by LC-MS-MRM in liver disease. J. Proteomics 2015, 116, 24−33.
F
DOI: 10.1021/acs.jproteome.5b00899 J. Proteome Res. XXXX, XXX, XXX−XXX
