MS Analysis of N-linked Glycans

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Article

Enhanced Quantitative LC-MS/MS Analysis of N-linked Glycans Derived from Glycoproteins Using Sodium Deoxycholate Detergent. Rui Zhu, Shiyue Zhou, Wenjing Peng, Yifan Huang, Parvin Mirzaei, Kaitlyn Donohoo, and Yehia Mechref J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00127 • Publication Date (Web): 10 May 2018 Downloaded from http://pubs.acs.org on May 12, 2018

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Journal of Proteome Research

Enhanced Quantitative LC-MS/MS Analysis of N-linked Glycans Derived from Glycoproteins Using Sodium Deoxycholate Detergent.

1 2 3 4 5 6 7 8

Rui Zhu1, Shiyue Zhou1, Wenjing Peng1, Yifan Huang1, Parvin Mirzaei1, Kaitlyn Donohoo1 and Yehia Mechref1*

9 10 11 12 13 14

1

Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX

15 16 17 18 19 20

Keywords: acidic labile detergent, sodium deoxycholate, detergent removal, N-glycomics, LCMS/MS sample preparation

21 22 23

*Corresponding Author

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Department of Chemistry and Biochemistry

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Texas Tech University

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Lubbock, TX 79409-1061

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Email: [email protected]

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Tel: 806-742-3059

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Fax: 806-742-1289

30

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ACS Paragon Plus Environment

Journal of Proteome Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

2

Protein glycosylation is a common protein post-translational modification (PTM) in living

3

organisms and has been shown to associate with multiple diseases, and thus may potentially be a

4

biomarker of such diseases. Efficient protein/glycoprotein extraction is a crucial step in the

5

preparation of N-glycans derived from glycoproteins prior to LC-MS analysis. Convenient,

6

efficient and unbiased sample preparation protocols are needed. Herein, we evaluated the use of

7

sodium deoxycholate (SDC) acidic labile detergent to release N-glycans of glycoproteins derived

8

from biological samples such as cancer cell lines. Compared to the filter-aided sample

9

preparation approach, the sodium deoxycholate (SDC) assisted approach was determined to be

10

more efficient and unbiased. SDC removal was determined to be more efficient when using

11

acidic precipitation rather than ethyl acetate phase transfer. Efficient extraction of

12

proteins/glycoproteins from biological samples was achieved by combining SDC lysis buffer and

13

beads beating cell disruption. This was suggested by a significant overall increase in the

14

intensities of N-glycans released from cancer cell lines. Additionally, the use of SDC approach

15

was also shown to be more reproducible than those methods that do not use SDC.

16

2


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Introduction

2

Protein glycosylation is a common and critical posttranslational modification (PTMs).1-4

3

Two types of glycans are defined based on the site of glycosylation: (i) N-glycans are covalently

4

attached to asparagine in an Asn-Xxx-Ser/Thr motif, where Xxx refers to any amino acids except

5

proline; and (ii) O-glycans are covalently linked to Ser/Thr residues.5 In mammals, protein

6

glycosylation plays essential roles in many biological systems, including cell recognition and

7

adhesion, protein stability and localization, and immune response.6-10 Moreover, recent studies

8

have reported that aberrant glycosylation is associated with a wide range of human diseases, such

9

as inflammatory diseases,11-13 rheumatoid arthritis,14, 15 neuronal injury16, 17 and cancer.18-21

10

Currently, the analysis of N-glycans derived from glycoproteins relies heavily on liquid

11

chromatography coupled to tandem mass spectrometry (LC-MS/MS).22 A typical protocol for

12

sample preparation involves several steps, including (i) the extraction of glycoproteins from cell

13

or tissue samples using physical stress (such as freeze/thaw cycles, beads-beating or ultrahigh-

14

frequency sonication); (ii) the release of N-glycans from extracted glycoproteins by Peptide-N-

15

glycosidase F (PNGase F); and (iii) the purification of released N-glycans by solid phase

16

extraction or lectin affinity chromatography.23-27 Released N-glycans are routinely derivatized or

17

directly analyzed by LC-MS/MS. Since detergent is commonly used to improve the protein

18

solubilization (many glycoproteins are hydrophobic membrane proteins) during the extraction

19

steps, and since it interferes with the potential glycan derivatization and down-stream MS

20

analysis, the purification steps are critical for the analysis of biological samples.28 However,

21

often times these purification steps increase the sample preparation time, sample loss and

22

variability of analytical measurements.

23

Several innovative approaches have been proposed to improve the sample preparation by 3



1

removing detergents effectively. Since the development of filter-aided sample preparation

2

(FASP), the well-known proteomics sample preparation method, filter devices have been used in

3

glycomic analysis.29-31 Combining filter devices and lectin affinity enrichment Zielinska et al.

4

proposed “N-glyco-FASP,” which requires the addition of lectin reagent into filter devices.29

5

Appling the chaotropic reagent based detergent removal strategy Rahman et al. developed

6

“Filter-Aided N‑Glycan Separation” (FANGS), which allows for the depletion of unwanted

7

detergent from the cell lysate.30 Incorporating trypsin and PNGase F digestion was introduced by

8

Zhou et al. “The GlycoFilter,” which is a comprehensive sample preparation platform for

9

proteomics, N-glycomics and glycosylation site assessment.31 These filter-aided sample

10

preparation methods utilized molecular weight cut-off filter devices to separate N-glycans from

11

small molecule detergents and deglycosylated proteins based on size differences. However, the

12

molecular cutoff of filters (10 kDa is popular) used in this protocol may cause some sample loss

13

and abundance bias when analyze glycoproteins whose sizes are similar (within ± 3 times of the

14

filter cutoffs).

15

An alternative way to avoid the interference from detergent in LC-MS/MS analysis is to

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solubilize glycoproteins using MS compatible detergents. The acid-labile surfactant RapiGest SF

17

(Waters) is able to enhance hydrophobic protein solubility and can be removed by acidification.32

18

More recently, sodium deoxycholate (SDC), a cheaper acid-labile detergent derived from

19

mammalian bile, has become a popular choice of MS compatible detergent in the field of

20

proteomics.33,

21

solubility, trypsin activity and can be easily removed by acidic precipitation or ethyl acetate

22

phase transfer. 35 Comparative studies have suggested that SDC based membrane protein sample

23

preparation protocol is the method of choice for unbiased and efficient protein solubilization.36, 37

34

Compared with traditional detergents, SDC significantly improves protein

4


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1

However, the performance of SDC in glycomics sample preparation is not investigated. This

2

might due to that the glycan purification includes not only detergent removal but also glycan

3

isolation from the deglycosylated proteins.

4

In this study, we evaluated several glycoprotein solubilizations and detergent removal

5

protocols for N-glycan analysis. Detergent-free protocols and protocols using different detergent

6

removal strategies are first compared using model glycoproteins. We also validated the use of

7

SDC in cell lysis buffers for biological sample preparation. Additionally, the influence of SDC

8

lysis buffer in different cell disruption methods is compared with detergent-free methods.

9

Material and Methods

10

Chemicals and reagents. Dithiothreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate

11

(ABC), sodium deoxycholate (SDC), Dimethyl sulfoxide (DMSO, >99.9%), urea, sodium

12

hydroxide beads (20-40 mesh), iodomethane, borane-ammonia complex, bovine ribonuclease B,

13

bovine fetuin and MS-grade formic acid were purchased from Sigma-Aldrich (St. Louis, MO).

14

High performance liquid chromatography (HPLC) grade acetonitrile, methanol, water, trypsin

15

EDTA 1X (0.25% Trypsin/2.21 mM EDTA) and 10mM phosphate-buffered saline (PBS, pH=

16

7.4) were purchased from Fisher Scientific (Pittsburgh, PA). Trypsin/Lys-C mix, mass

17

spectrometry grade was obtained from Promega (Madison, WI) and PNGase F (Glycerol-free,

18

500,000 units/ml) from New England Biolab (Ipswich, MA). iGlycoMab was provided by

19

GlycoScientific (Athens, GA).

20

Sample preparation of model glycoproteins. All experiments were performed in triplicates. The

21

summary of protocols compared in this study is shown in Table 1. Bovine ribonuclease B,

22

bovine fetuin and iGlycoMab IgG were prepared in 10mM PBS buffer (pH= 7.4) with the final

23

concentrations of 2.5 µg/µL to make a stock model glycoprotein solution. For in-solution sample 5



1

preparation protocols, including in solution digestion of proteins extracted by PBS (IS-PBS), in

2

solution digestion of proteins extracted by SDC and acid precipitation (IS-SDC-AP) and in

3

solution digestion of proteins extracted by SDC and phase transfer (IS-SDC-PT), 5-µL aliquots

4

of stock model glycoprotein solutions were mixed with 5 µL of 10mM PBS buffer or SDC

5

solution (5% w/v SDC in 10mM PBS, pH = 7.4). The mixtures were further diluted with another

6

40 µL of 10mM PBS buffer (to make the final concentration of proteins 0.25 µg/µL and SDC 0.5%

7

w/v) and thermally denatured at 90 oC for 10 mins. Next, about 50 units of PNGase F were added

8

to the denatured samples. Then the samples were incubated for 18 hours at 37 oC. After the

9

PNGase F digestion, SDC was removed by adding 1% v/v neat formic acid and centrifuging (IS-

10

SDC-AP) or following the standard phase transfer protocol as previously described (IS-SDC-

11

PT).35 For standard phase transfer protocol, briefly, equal volume of ethyl acetate was added to

12

the digested sample. Then the mixture was acidified with FA (0.5% as final concentration),

13

shaken thoroughly for 1 min and centrifuged at the maximum speed for 2 min. The aqueous

14

phase was collected.

15

Glycans were further purified by protein precipitation and dialysis. Protein was precipitated

16

by 90% ice-cold ethanol aqueous solution, and excessive salts were removed by a home-made

17

dialysis device fitted with 500-1000 Da MWCO dialysis membrane. The purified glycans were

18

vacuum dried and stored at -20 oC before reduction and permethylation.

19

For the in filter sample preparation protocols, including in filter digestion of proteins

20

extracted by PBS (IF-PBS), in filter digestion of proteins extracted by SDS (IF-SDS) and in filter

21

digestion of proteins extracted by SDC (IF-SDC), 5-µL aliquots of stock model glycoprotein

22

solutions were mixed with 5 µL of 10mM PBS buffer, SDC solution (5% w/v SDC in 10mM

23

PBS, pH = 7.4) or 5% SDS solution (5% w/v SDS in 10mM PBS, pH = 7.4). The mixtures were 6


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1

further diluted with 340 µL of urea solution (8M urea in 10mM PBS buffer, pH = 7.4). Next, the

2

samples were subjected to filter-aided N‑glycan separation (FANGS).30 Briefly, samples were

3

transferred to an ultrafiltration device (Amicon Ultra-0.5, Ultracel-30 membrane, nominal mass

4

cutoff 10 kDa, Millipore) and centrifuged at 14, 000g for 10 min. The samples were washed

5

twice with 300 µL of urea solution and centrifugation at 14, 000g for 10 min. Four additional

6

wash steps were performed by adding 300 µL of 10mM PBS buffer (pH= 7.4) followed by

7

centrifugation. Next, about 50 units of PNGase F were added to the samples. The filter devices

8

were sealed with Parafilm and incubated for 18 hours at 37 oC. After the digestion, the devices

9

were centrifuged at 14, 000g for 10 min, and three times they were washed with 300 µL of

10

HPLC water and centrifuged. The released N-glycans were vacuum dried for future reduction

11

and permethylation.

12

Cell cultivation, harvest and counting. MDA-MB-361 was grown with RPIM-1640 medium

13

(with 20% Fetal Bovine Serum and 2% Penicillin-Streptomycin Solution) in 175 cm2 flasks until

14

80% cell confluence was reached. Cell number was counted by a hemocytometer (Thermo

15

Scientific, San Jose, CA) and then cell concentration was determined as follows: cell

16

concentration = total cell number on the counting area/4×104 cell/mL. Cells were washed twice

17

with 10mM PBS buffer (pH= 7.4). After washing, the cell pellet was resuspended in 10mM PBS

18

buffer (pH=7.4), and samples were evenly divided (~5 million cells per tube) into aliquots for

19

different protocol tests.

20

Cell lysis protocols. The PBS buffer was removed by centrifugation. Aliquots of ~5 million of

21

cells were resuspended in 100 µL of 10mM PBS buffer (pH=7.4) or SDC solution (5% w/v SDC

22

in 10mM PBS, pH = 7.4). Three cell lysis techniques were compared: beads beating, freeze/thaw

23

cycles and ultrahigh frequency sonication. Each method was performed in triplicates. For beads 7



1

beating, the samples were homogenized using a Beadbug microtube homogenizer (Benchmark

2

Scientific, Edison, NJ). Briefly, ~100 µL of triple high impact zirconium beads (Ø: 0.15 mm)

3

were mixed with each sample in a 2-mL microtube. Homogenization was performed three times

4

at 40k rpm for 90 seconds with a 30-second rest in between. For freeze/thaw cycles, three

5

freeze/thaw cycles were performed on each sample. Each freeze/thaw cycle was conducted by

6

incubating at room temperature (25 °C) for 30 minutes and returning to −20 °C for 1 hour. For

7

ultrahigh frequency sonication, an additional 150-µL aliquot of 10mM PBS buffer (pH=7.4) or

8

SDC solution (5% w/v SDC in 10mM PBS, pH = 7.4) were added to each sample. The samples

9

were lysed using an ultrasonic processor (Cole-Parmer Instruments, Vernon Hills, IL) equipped

10

with a 3mm sonication probe for 8 cycles (25 sec of sonication at 30% power, followed by 20

11

sec of incubation on ice).

12

Trypsin and PNGase F treatment of whole cell lysate. A 100-µL aliquot of each sample of

13

whole cell lysate was diluted to 10 times of the original concentration with 50mM ammonium

14

bicarbonate buffer (pH=7.8), and the rest was diluted 10 times of the original concentration with

15

10mM PBS buffer (pH=7.4). Next, the samples were thermally denatured at 90 oC for 10 mins.

16

The extracted protein concentration was determined by BCA protein assay (Thermo Scientific,

17

San Jose, CA). Each sample was reduced by adding DTT to a final concentration of 5mM and

18

incubated for 45 min at 60 oC. The mixture was alkylated by adding IAA to a final concentration

19

of 20 mM and incubated in the dark for 45 min. The reaction was quenched with another

20

addition of 5 mM DTT. Tryptic digestion was performed by adding the Trypsin/Lys-C mixture

21

(Promega, Madison, WI) using a 1:25 enzyme to protein ratio and incubated at 37.5 oC for 18

22

hours. The digestion was quenched, and the SDC was precipitated by adding 1% (v/v) neat

23

formic acid. The mixture was centrifuged at 21,000 g for 10 min. The supernatant was collected, 8


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1

vacuum dried and kept at -20 oC. The samples were resuspended in solvent A (2% ACN and

2

0.1% formic acid in HPLC water) immediately before LC-MS injection. PNGase F digestion was

3

performed on the reduced and alkylated samples as described above in the in solution sample

4

preparation protocol.

5

Reduction and permethylation of released N-glycans. Glycans released from model

6

glycoproteins and biological samples were reduced and then permethylated following the

7

protocol that has been previously reported in many papers.25,

8

reduced by 10 µl borane ammonia complex solution (10 mg/mL) at 60oC for 1 h. Then the

9

remaining borate was removed by adding 500 µl methanol and drying down for 3 times. Reduced

10

glycans were resuspended in 30 µl DMSO, 1.2 µl water and 20 µl iodomethane and then loaded

11

in the spin column which contained sodium hydroxyl beads (stored in DMSO). After sample

12

loading, the column was incubated at room temperature for 25 min. After 25 min, 20 µl of

13

iodomethane was added to the column and incubated for another 15 min. Then the sample was

14

collected by centrifuging at 1800 rpm for 2 min and dried overnight. After completely dried,

15

reduced and permethylated glycans were ready to use.

16

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. LC-MS/MS

17

analysis was performed using a Dionex 3000 Ultimate nano-LC system (Thermo Scientific, San

18

Jose, CA) interfaced to LTQ Orbitrap Velos (Thermo Scientific, San Jose, CA) equipped with a

19

nano-ESI source. The online purification was performed using a C18 Acclaim PepMap 100

20

trapping column (75 µm I.D. x 2 cm, 3 µm particle sizes, 100 Å pore sizes, Thermo Scientific,

21

San Jose, CA), and the separation was attained using a C18 Acclaim PepMap RSLC column (75

22

µm I.D. x 15 cm, 2 µm particle sizes, 100 Å pore sizes, Thermo Scientific, San Jose, CA).

9


38-42

Generally, glycans were


1

A 60-minute LC method was used for the separation of permethylated glycans. For the

2

analysis of N-glycans derived from model glycoproteins, 1/10 of N-glycans derived from 12.5µg

3

bovine ribonuclease B and 12.5µg bovine fetuin was used for each injection. For the analysis of

4

N-glycans derived from cell lysate, N-glycans released from half of a million cells or 50µg of

5

protein were used for each injection. During the separation, the column compartment was

6

maintained at 55 oC. The LC elution gradient of solvent B was: 20% solvent B over 10 min,

7

20%-42% over 1 min, 42%-55% over 27 min, 55%-90% over 1 min, 90% over 4 min, 90%-20%

8

over 1 min and 20% over 5min. Solvent B consisted of 100% ACN with 0.1% formic acid while

9

solvent A consisted of 2% ACN with 0.1% formic acid added to HPLC grade water. The LTQ

10

Orbitrap Velos mass spectrometer was operated in the positive ion mode with the ESI voltage set

11

to 1.6kV. Full MS was operated at 15,000 resolution, and the scan range was set to 700-2000

12

m/z. MS2 was conducted in data-dependent acquisition (DDA) mode. The top 4 intensity

13

precursor ions from the full scan were subjected to both CID and HCD. Normalized collision

14

energy (CE) was set to 30% and 45% for CID and HCD, respectively. The dynamic exclusion

15

was set to have a repeat count of 2, repeat duration of 30s, exclusion list size of 50 and exclusion

16

duration of 60s. Precursor ion selection window was set to 1.50, intensity threshold for MS2 was

17

5000 counts, singly charged ions were set to be excluded for MS2.

18

A 120-minute LC method was used for the separation of protein digest. One µg of protein

19

digest was used for each injection. During the separation, the column compartment was

20

maintained at 29.5 oC. The LC elution gradient of solvent B used in both LC-MS/MS analysis

21

was: 5% over 10 min, 5%-20% over 55 min, 20-30% over 25 min, 30-50% over 20 min, 50%-

22

80% over 1 min, 80% over 4 min, 80%-5% over 1 min and 5% over 4 min. Data-dependent

23

acquisition mode was employed to achieve two scan events. The first scan event was a full MS 10


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1

scan of 400-2000 m/z at a mass resolution of 15,000. The second scan event was CID MS/MS of

2

precursor ions selected from the first scan event at normalized collision energy (CE) of 35%. The

3

CID MS/MS scans were performed on the ten most intense ions observed in the MS scan event.

4

The dynamic exclusion was set to have a repeat count of 2, repeat duration of 30s, exclusion list

5

size 200 and exclusion duration of 90s. Precursor ion selection window was set to 0.5, intensity

6

threshold for MS2 was 5000 counts, singly charged ions were set to be excluded for MS2.

7

Data Analysis. The glycomics data was processed by MultiGlycan43, 44 and verified manually by

8

calculating the peak areas of extracted ion chromatograms (EIC). The search parameters of

9

MultiGlycan were set as follow: the mass tolerance was set to 5 ppm; isotope envelope tolerance

10

was set to 15 ppm, and a minimum number of peaks in a cluster were set to 3. Peaks

11

corresponding to glycans in different adduct forms and charge states were summed up to

12

represent the intensity of each glycan. For the N-glycans derived from cell lysate, the absolute

13

values of the intensities were divided by the intensity of 15N HexNAc4Hex3dHex1 (which is the

14

most abundant glycan derived from iGlycoMab IgG) to represent the normalized intensity of

15

each glycan.45

16

The proteomic LC-MS/MS raw data was converted to mascot generic format files (*.mgf)

17

by Proteome Discover version 1.2 software (Thermo Scientific, San Jose, CA) then searched

18

using UniProt database database (2014_06, Homo sapiens, 20214 entries) in MASCOT version

19

2.4 (Matrix Science Inc., Boston, MA). Carbamidomethylation of cysteine was set as a fixed

20

modification while oxidation of methionine was set as a variable modification. The m/z tolerance

21

of MS and MS/MS were set to 6 ppm and 0.8 Da, respectively. Maximum peptide missed

22

cleavages was set to 2. MASCOT search result was validated using Scaffold (version

23

Scaffold_3.6.3, Proteome Software Inc., Portland, OR). Peptide identifications were accepted at 11



Page 12 of 34

1

higher than 95.0% probability as specified by the Peptide Prophet algorithm.46 Protein

2

identifications were accepted if they could be established at higher than 99.0% probability and

3

contained at least 2 identified peptides. Protein probabilities were assigned by the Protein

4

Prophet algorithm.47 Proteins that contained similar peptides and could not be differentiated

5

based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Membrane

6

proteins were annotated with gene ontology (GO) terms from NCBI.48 The mass spectrometry

7

proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner

8

repository with the dataset identifier PXD008687 (Reviewer account details: Username:

9

[email protected] Password: uJb06RfG).49

10

Result and discussion

11

Filter-aided sample preparation methods have been successfully employed in glycomics

12

sample preparation of biological samples.29, 30, 50 However, the size of some small glycoproteins,

13

e.g., bovine ribonuclease B (RNase B, ~16kDa), is close to the cut-off of the commonly used 10

14

kDa molecular weight cut-off filter and the investigation of sample loss and abundance bias is

15

scarce. On the other hand, SDC has been used as a useful acid labile detergent that can be

16

removed by acidic precipitation or ethyl acetate phase transfer in the field of proteomics for

17

years, but its influence on glycomics sample preparation remained unknown.33, 35 Even in the

18

proteomics field, opinions differ on the choice of the most efficient and unbiased removal

19

protocols.35-37,

20

method since the acidic precipitation will potentially co-precipitate hydrophobic peptides from

21

the solution and lead to a bias in the proteome coverage.

22

precipitation is a better choice since it is easier and safer experiment procedure and the

23

proteomics profile of the two shows no marked differences.37, 51 As shown in Figure 1a, we first

51

Some studies have claimed ethyl acetate phase transfer is a slightly better

35, 36

12


Other studies insist that acidic

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1

compared different protocols by using two model glycoproteins: bovine ribonuclease B (RNase

2

B), a well-studied glycoprotein contains exclusively high mannose oligosaccharides,52 and

3

bovine fetuin, another well-studied glycoprotein contains triantennary and biantennary type

4

sialylated glycans53 to assess sample loss and bias of filter aided method and validate the use of

5

SDC in glycomics analysis. As shown in Figure 1b, we compared the performance of SDC lysis

6

buffer and detergent free lysis buffer in three commonly used physical cell disruption techniques:

7

beads beating, freeze/thaw cycles and ultrahigh frequency sonication.

8

Evaluation of sample loss and abundance bias using model glycoproteins

9

Figure 2a depicts the total N-glycan yield of all six compared protocols (Figure 1a). The

10

total peak areas (acquired by adding up all absolute peak areas of individual glycans) of high-

11

mannose and sialylated type glycans represented the N-glycans yield of RNase B and fetuin,

12

respectively. Significant sample loss was observed in the filter aided protocols, Figure 2a.

13

Despite the fact that different solutions (PBS or SDC) were used to dissolve the model

14

glycoproteins, the sample losses in the filter aided protocols were consistently around 50% of

15

high mannose type glycans (IF-PBS: IS-PBS= 0.47:1, IF-SDC: IS-SDC-AP= 0.45:1) and 65% of

16

sialylated glycans (IF-PBS: IS-PBS= 0.34:1, IF-SDC: IS-SDC-AP= 0.35:1). This variation

17

observation might be due to the filter surface, which consists of cellulose. Studies have shown

18

powder and cotton cellulose enrich glycopeptides via hydrophilic interaction between the

19

hydroxyl groups of both cellulose and glycan moieties.54, 55 Relative to glycopeptides, glycans

20

are more hydrophilic and expected to efficiently interact with abundant hydroxyl groups of

21

cellulose through hydrophilic interaction. Moreover, additional sample handling associated with

22

filter-aided protocol might be an additional reason for sample loss. Although the IF-SDS

23

protocol performed slightly better than the PBS control and SDC-filter protocols, the sample loss 13



1

is still close to 50% of high mannose type glycans (IF-SDS: IS-SDC-AP=0.52:1), and 60% of

2

sialylated glycans (IF-SDS: IS-SDC-AP=0.40:1).

3

The comparison of detergent free and SDC methods suggested that the use of SDC

4

improves the glycan yield, Figure 2a. Although SDC was not expected to improve the solubility

5

of the model glycoproteins which are soluble in PBS buffer at low concentrations, the SDC-AP

6

method allows ~45% more glycan yield from RNase B (SDC-AP: PBS= 1.45: 1, SDC-filter:

7

PBS-filter= 1.43: 1) and >80% from fetuin (SDC-AP: PBS= 1.84: 1, SDC-filter: PBS-filter=

8

1.91: 1). Accordingly, the digestion efficiency of model glycoproteins denatured in water at 90oC

9

and incubated overnight with PNGase F still could be improved by SDC. Since SDC is known to

10

improve the accessibility of trypsin to the substrate and enhance the trypsin activity, 35 a similar

11

mechanism could also apply to PNGase F digestion: denatured glycoproteins are more accessible

12

in SDC solutions.

13

A marked difference between the two SDC removal methods (IS-SDC-AP and IS-SDC-PT)

14

were observed in this study, Figure 2a. As mentioned above, the acidic precipitation procedure

15

was suspected to co-precipitate hydrophobic peptides but not hydrophilic glycans, while the

16

addition of ethyl acetate would induce protein precipitation that increases the difficulty of

17

pipetting in the microcentrifuge tube. These two factors might have led to more sample loss in

18

the SDC-PT protocol. However, the loss of high mannose and sialylated type glycans were not

19

comparable. SDC-PT resulted in depleting high mannose glycans selectively from the sample.

20

For high mannose glycans, the ratio between methods IS-SDC-PT and IS-SDC-AP was about to

21

0.19:1; and for sialylated species, the ratio increased to 0.60:1. At this point, we don't have a

22

conclusive explanation for this phenomenon. A possible explanation is that ethyl acetate

23

dissolves more high-mannose glycans, which are relatively hydrophilic, and less sialylated 14


Page 14 of 34

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1


glycans, which are relatively hydrophobic at the pH used here.

2

We also compared the protocols using the relative abundance (acquired through dividing

3

abundance of individual glycan by total glycan abundance) individual N-glycans (Figure 2b and

4

c), to assess the abundance bias of different sample preparation protocols. Two distinct relative

5

abundance distribution patterns of N-glycans derived from RNase B were observed, Figure 2b.

6

Compared with filter aided protocols, in solution protocols resulted in a higher abundance of

7

Man5GlcNAc2 and a lower abundance of Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2. The

8

classic H NMR demonstrated an relative abundance of 57%, 31%, 1.5%, 1.5%, and 1.0% for

9

Man5GlcNAc2, Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 glycans.52 Since

10

the distributions detected from in solution protocol samples were more similar to the H NMR

11

results, the sample loss of the filter aided methods has been discussed above, we believe the filter

12

devices created this bias. This might be due to the molecular weight of RNase B (~17.6 kDa with

13

Man5GlcNAc2 and ~18.3k Da with Man9GlcNAc2) is close to the filter cut-off weight (10 kDa).

14

According to manufacture’s specification, filter cutoff is not absolute. Whether a protein can

15

pass through the filter not only depends on its molecular weight, but also depends on its shape

16

(rod or sphere), flexibility and hydrodynamic volume, etc. Usually, to guarantee none sample

17

loss, protein’s molecular weight should be 3 times higher than filter molecular weight cutoff

18

(MWCO)

19

https://www.sigmaaldrich.com/technical-documents/articles/biology/amicon-ultra-centrifugal-

20

filters.html). Therefore, when we used 10kDa filter, the safe molecular weight of protein which

21

can be 100% retained on the top should be 30 kDa. And the molecular weight of RNase B was so

22

close to the filter cutoff that there was a possibility of sample loss. Hence, the proteoforms with

23

smaller glycan structures lost more sample during the sample preparation than those with more

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1

extensive glycan structures. The relative distribution of N-glycans derived from fetuin also

2

supported this statement, Figure 2c. The abundance distributions detected from all protocols

3

were roughly in the same level. This might be due to the larger size of fetuin (~38 kDa)

4

prohibiting selective sample loss. On the other hand, SDC-PT protocol generated a slightly

5

different pattern with higher abundances of tri- and tetra-sialylated glycans and lower

6

abundances of mono- and bi-sialylated glycans. This is consistent with the aforementioned

7

results that ethyl acetate seems to remove more hydrophilic glycans than hydrophobic ones. In

8

addition, the observed bias in IF protocol might also be induced by different denaturation

9

methods (urea denaturation in IF vs. thermal denaturation in IS) and different glycan structures,

10

thus causing different PNGase F efficiency.56 However, there are several reports that showed

11

thermal denaturation in water was equal or less effective than urea assisted denaturation of

12

protein.57,

13

denaturation) should not be better than that in in-filter (IF) protocols (urea denaturation).

14

Therefore, the IS protocol (thermal denaturation) was better than IF protocol (urea denaturation)

15

was not becauce of the different denaturation methods used here. Since each comparison used

16

the same glycoproteins, the glycan structures were the same, that could not prompt a PNGase F

17

treatment bias. Thus we believe it is more reasonable that the distribution bias in RNase B might

18

due to filter device as discussed above.

58

Thus, PNGase F digestion efficiency in in-solution (IS) protocols (thermal

19

To summarize, we investigated the sample loss and bias of detergent free, SDC based and

20

filter-aided sample preparation methods for N-glycan analysis. The most efficient and unbiased

21

approach was determined to be the use of 0.5% SDC in the digestion buffer, and that is removed

22

by acidic precipitation at the conclusion of digestion. Significant sample losses and abundance

23

biases were observed in IS-SDC-PT and all filter aided methods. Therefore, these methods were 16


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1

deemed unreliable and not suited for the preparation of biological samples. Detergent-free and

2

IS-SDC-AP methods were further evaluated in biological sample preparation.

3

N-glycan released from biological samples

4

Detergent-free (PBS) or IS-SDC-AP protocols were combined with three cell disruption

5

techniques, including beads beating, freeze/thaw cycles and ultrahigh frequency sonication, to

6

determine the optimal N-glycan sample preparation methods of biological samples. Protein

7

assays and proteomics profiling were used to evaluate the protein extraction efficiency. As

8

shown in Figure 3a, SDC lysis buffer enhanced the overall protein solubility of a whole cell

9

lysate of ~5 million MDA-MB-361 cells. The combination of physical disruptions with SDC

10

lysis buffer resulted in the extraction of approximately the same proteome amount. However,

11

beads beating and freeze/thaw cycles performed better than ultrahigh frequency sonication as

12

suggested by the variability of extraction (i.e., smaller standard deviations). The highly viscous

13

SDC lysis buffer resulted in the attachment of the metal probe of ultrahigh frequency sonication,

14

thus introducing additional variance to the SDC-UHS method. Unlike SDC lysis buffer, the

15

detergent-free method resulted in different levels of protein extraction when different disruption

16

methods were used. This might be due to the physical stress that induces protein aggregation and

17

denaturation via distinct mechanisms, and hence led to the solubility variance observed.

18

Membrane proteomics profile also demonstrated the SDC lysis buffer outperformed detergent-

19

free lysis buffer when the disruption methods were not critical factors in cell lysis (Figure 3b

20

and Figure S-1a). Details of quantitative proteomic results were shown in Supporting

21

Information Table S-1. Spectral counts of each protein, which is the total number of spectra

22

identified for a protein, were initially used for protein quantitation of each sample. To better

23

compare among different samples, a normalized spectral count was applied. It was achieved 17



1

through normalizing each protein abundance in one sample by the total protein abundances

2

across all samples. Venn graph of identified proteins shown in Figure S-1a indicates the

3

proteomics profile of all SDC methods were more consistent than those detergent-free methods.

4

The three cell lysis approach which used SDC permitted 998 proteins in common while SDC-

5

free methods only had 757 overlapped proteins. Similar to previously published studies, the

6

proteomics profile suggested that SDC significantly enhances the solubility of membrane

7

proteins.36, 37 Although the SDC improved only the overall protein identifications by 10~20%;

8

the additionally identified proteins were membrane proteins (Figure S-1b). The number of

9

identified membrane proteins increased in both three SDC methods relative to SDC-free methods.

10

In addition, gene ontology enrichment (Figure S-1c) also confirmed the increase of membrane

11

proteins when using SDC, which could contribute to the glycomics studies. Accordingly, the

12

addition of SDC to the lysis buffer played a significant role in improving the solubility of

13

hydrophobic proteins, thus improving total glycome.

14

The number of identified N-glycans derived from the same number of MDA-MB-361 cells

15

(~5 million cells) using different extraction protocols is shown in Figure 4, while the total

16

normalized glycan yields of the different methods used in the study are shown in Figure S-2.

17

Supporting Information Table S-2 depicts the identification of glycan structures in this study,

18

including theoretical m/z, observed m/z, mass accuracy (deviation) of each replicate. The

19

average of mass accuracy of all glycan structures in this study was less than 1 ppm, which

20

indicated a reliable glycomics analysis using our instruments without a lock-mass. Figure S-3

21

depicts an example of total ion chromatogram (TIC) of glycomics analysis using LC-MS/MS and

22

corresponding mass spectra with designated glycan structures. Three areas in TIC (blue areas)

23

were zoomed in as examples to show the identification of glycan structures, as shown in Figure 18


Page 18 of 34

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1

S-3a, 3b and 3c. Similar to the proteomic results, SDC lysis buffer based protocols outperformed

2

the detergent-free methods in both qualitative and quantitative aspects while the cell disruption

3

step was less critical. As shown in Figure 4a-c, the LC-MS analysis of permethylated glycans

4

allowed the identification of 58 N-glycan in all SDC protocols, while only 47, 48 and 44 glycans

5

were identified using PBS-BB, PBS-F/T and PBS-UHS protocols, respectively. There were 11,

6

10 and 14 more glycan structures identified in SDC protocols when compared with relative PBS

7

protocols. Quantitatively, the total N-glycan yield data (Figure S-2) exhibited a similar trend

8

with the protein assay results: all SDC based methods released more N-glycans from the whole

9

cell lysate while the detergent-free methods produced lower numbers of N-glycans. For each N-

10

glycan, a quantitative comparison was performed by calculating the log2 transformed ratio

11

between the SDC lysis buffer and detergent free results (Figure 4d-f). The result shows that N-

12

glycan intensities using SDC lysis buffer are on average 3.53-, 1.17- and 14.82-times more

13

abundant than detergent-free lysis buffer when combined with beads beating, freeze/thaw cycles

14

and ultrahigh sonication cell disruption, respectively. The full list of identified N-glycans and

15

their intensities (normalized by the internal standard

16

listed in Table S-3.

15

N labeled HexNAc4Hex3DeoxyHex1) are

17

The protocol comparison of selected N-glycans released from the same amount of protein

18

(50µg) derived from MDA-MB-361 cells is depicted in Figure 5. Peak area data are normalized

19

by the total intensity to represent the relative abundance of the individual N-glycan composition.

20

Figure 5a depicts the abundance distribution of high-mannose type glycans, which were the

21

most abundant N-glycans in the cell samples. The relative distributions of Man6GlcNAc2,

22

Man8GlcNAc2, and Man9GlcNAc2 detected from SDC based protocols were higher than those

23

observed in detergent free based protocols. For both SDC and detergent free methods, lower 19



1

abundances of Man5GlcNAc2 were observed in the samples extracted by ultrahigh frequency

2

sonication. On the other hand, the analytical variabilities of sample preparation methods were

3

mainly associated with the cell disruption methods (average relative standard deviation: PBS-

4

BB= 6.31%, SDC-BB= 8.12%, PBS-F/T= 14.29%, SDC-F/T=15.69%, PBS-UHS=17.94%,

5

SDC-UHS:12.86%). Among all three compared cell disruption methods, beads beating was the

6

most reproducible method. However, lower than 20% relative standard deviations were observed

7

for all protocols, which represents approximately reproducible results for LC-MS quantitation.

8

Figure 5b depicts the abundance distribution of triantennary type glycans, which are

9

relatively low abundant glycans derived from cell samples. We took triantennary structures as an

10

example because they were more representative (we identified almost all triantennary structures

11

with different number of sialic acid) in low abundant glycans. Two distinct distribution patterns

12

could be observed between SDC based protocols and detergent free protocols. For example, in

13

SDC based protocols, HexNAc6Hex5Fuc1NeuAc3 was lower in abundance while HexNAc6Hex5

14

and HexNAc6Hex5NeuAc1 were higher in abundance than their counterparts in detergent free

15

protocols. Another finding was that the reproducibility of detergent free protocols was poor for

16

these low abundant structures. This variation might be due to the poor solubility of membrane

17

proteins in detergent-free lysis buffer and that the low abundant N-glycans are mostly released

18

from membrane proteins. The full list of detected N-glycans and their relative abundances are

19

summarized in Table S-4. Relative abundance was acquired through dividing absolute

20

abundance of each individual glycan by the total abundance of all glycans.

21

According to the above-described result, SDC appears to be a valuable choice of detergent

22

to achieve efficient protein extraction and subsequent N-glycan release. Although beads beating

23

cell lysis performs slightly more efficient than the other two disruption methods, the addition of 20


Page 20 of 34

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1

SDC appeared to be more critical to achieve high efficiency and unbiased N-glycan release,

2

assuming that all other variables associated with the sample preparation are maintained constant.

3

Conclusion

4

Here, we validated the application of SDC in N-glycan sample preparation for LC-MS/MS

5

analysis. Relative to the detergent-free solubilization and the well-established filter-aided sample

6

preparation methods, the SDC based method was more efficient, convenient and unbiased.

7

Unlike proteomic sample preparation, detergent removal using acid precipitation outperformed

8

ethyl acetate phase transfer in the N-glycan analysis, causing less sample loss and bias to

9

different N-glycans. The combination of SDC lysis buffer and physical cell disruption methods

10

improved the solubility of membrane proteins and further enhanced the whole cell glycomic

11

analysis both qualitatively and quantitatively. Among the three evaluated cell disruption methods,

12

beads beating appears to be the most compatible with SDC lysis buffer. The corresponding

13

protocol enables the accurate and reproducible glycomics studies of a variety of biological

14

samples such as cell lines, blood serum and tissue lysate.

15 16

Author Contributions

17

The manuscript was written through contributions of all authors. / All authors have given

18

approval to the final version of the manuscript.

19

Acknowledgement

20 21 22 23 24

This work was supported by NIH grant (1R01GM112490-03) and CPRIT (RP130624).

Conflict of interest statement The authors declare no conflict of interest. SUPPORTING INFORMATION 21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Table S-1. Proteome quantitation of cell line MDA-MB-361 using different sample preparation protocols. (Excel file) Table S-2. Summary of identification masses of glycan structures derived from MDA-MB-361 cells using beads beating (BB), freeze and thaw cycles (F/T) and ultrahigh sonication (UHS). (Excel file) Table S-3. Full list of normalized intensities of N-glycans derived from same number of MDAMB-361 cells using beads beating, freeze and thaw cycles and ultrahigh sonication cell disruption in SDC lysis buffer and detergent free lysis buffer. (pp. 3-5) Table S-4. Full list of relative abundances of N-glycans derived from same amount of protein derived from MDA-MB-361 cells using beads beating, freeze and thaw cycles and ultrahigh sonication cell disruption in SDC lysis buffer and detergent free lysis buffer. (pp. 6-8) Figure S-1. Proteomics profile of MDA-MB-361 whole cell lysate extracted by different protocols. a. Venn graph of all identified proteins indicated the proteomics profile of all SDC methods were similar. b. Bar graph of total identified number of membrane proteins and nonmembrane proteins (other protein) from all three replicates demonstrated the major discrepancy of SDC and detergent free methods was contributed by membrane proteins. c. Gene ontology of proteome extracted from MDA-MB-361. The major difference of SDC and detergent free methods were highlighted. (p. 9) Figure S-2. Total N-glycan yields from ~5 million MDa-MB-361 cells. The total N-glycan yield was calculated by adding 15N standard normalized peak areas of each N-glycan structure. (p. 10) Figure S-3. Total ion chromatogram (TIC) and corresponded mass spectra with designated glycan structures from cell line MDA-MB-361. a, b and c represent average mass spectra from specific areas (blue areas) in TIC. Red areas in a and c denote the zoom in areas. (p. 11)

22


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References (1) Apweiler, R.; Hermjakob, H.; Sharon, N., On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochim Biophys Acta. 1999, 1473, (1), 4-8. (2) Wooding, K. M.; Peng, W.; Mechref, Y., Characterization of Pharmaceutical IgG and Biosimilars Using Miniaturized Platforms and LC-MS/MS. Curr Pharm Biotechnol. 2016, 17, (9), 788-801. (3) Mayampurath, A. M.; Wu, Y.; Segu, Z. M.; Mechref, Y.; Tang, H., Improving confidence in detection and characterization of protein N-glycosylation sites and microheterogeneity. Rapid Commun Mass Spectrom. 2011, 25, (14), 2007-2019. (4) Mayampurath, A.; Yu, C. Y.; Song, E.; Balan, J.; Mechref, Y.; Tang, H., Computational framework for identification of intact glycopeptides in complex samples. Anal Chem. 2014, 86, (1), 453-463. (5) Zhu, R.; Zacharias, L.; Wooding, K. M.; Peng, W.; Mechref, Y., Glycoprotein Enrichment Analytical Techniques: Advantages and Disadvantages. Methods Enzymol. 2017, 585, 397-429. (6) Bucior, I.; Scheuring, S.; Engel, A.; Burger, M. M., Carbohydrate-carbohydrate interaction provides adhesion force and specificity for cellular recognition. J Cell Biol. 2004, 165, (4), 529-537. (7) Helenius, A.; Aebi, M., Intracellular functions of N-linked glycans. Science. 2001, 291, (5512), 2364-2369. (8) Varki, A., Biological roles of oligosaccharides: all of the theories are correct. Glycobiology. 1993, 3, (2), 97-130. (9) O'Connor, S. E.; Imperiali, B., Modulation of protein structure and function by asparagine-linked glycosylation. Chem Biol. 1996, 3, (10), 803-812. (10) Veillon, L.; Huang, Y.; Peng, W.; Dong, X.; Cho, B. G.; Mechref, Y., Characterization of isomeric glycan structures by LC-MS/MS. Electrophoresis. 2017, 32, (17), 2100-2114. (11) Campbell, B. J.; Yu, L. G.; Rhodes, J. M., Altered glycosylation in inflammatory bowel disease: a possible role in cancer development. Glycoconj J. 2001, 18, (11-12), 851-858. (12) Dube, D. H.; Bertozzi, C. R., Glycans in cancer and inflammation--potential for therapeutics and diagnostics. Nat Rev Drug Discov. 2005, 4, (6), 477-88. (13) Song, E.; Zhu, R.; Hammoud, Z. T.; Mechref, Y., LC-MS/MS quantitation of esophagus disease blood serum glycoproteins by enrichment with hydrazide chemistry and lectin affinity chromatography. J Proteome Res. 2014, 13, (11), 4808-4820. (14) Elliott, M. A.; Elliott, H. G.; Gallagher, K.; McGuire, J.; Field, M.; Smith, K. D., Investigation into the concanavalin A reactivity, fucosylation and oligosaccharide microheterogeneity of alpha 1-acid glycoprotein expressed in the sera of patients with rheumatoid arthritis. J Chromatogr B Biomed Sci Appl. 1997, 688, (2), 229-237. (15) Smith, K. D.; Pollacchi, A.; Field, M.; Watson, J., The heterogeneity of the glycosylation of alpha1-acid glycoprotein between the sera and synovial fluid in rheumatoid arthritis. Biomed Chromatogr. 2002, 16, (4), 261-266. (16) Abou-Abbass, H.; Abou-El-Hassan, H.; Bahmad, H.; Zibara, K.; Zebian, A.; Youssef, R.; Ismail, J.; Zhu, R.; Zhou, S.; Dong, X.; Nasser, M.; Bahmad, M.; Darwish, H.; Mechref, Y.; Kobeissy, F., Glycosylation and other PTMs alterations in neurodegenerative diseases: Current status and future role in neurotrauma. Electrophoresis. 2016, 37, (11), 1549-1561. (17) Abou-Abbass, H.; Bahmad, H.; Abou-El-Hassan, H.; Zhu, R.; Zhou, S.; Dong, X.; Hamade, E.; Mallah, K.; Zebian, A.; Ramadan, N.; Mondello, S.; Fares, J.; Comair, Y.; Atweh, S.; Darwish, H.; Zibara, K.; Mechref, Y.; Kobeissy, F., Deciphering glycomics and neuroproteomic alterations in experimental traumatic brain injury: Comparative analysis of aspirin and clopidogrel treatment. Electrophoresis. 2016, 37, (11), 1562-1576. (18) Mechref, Y.; Hu, Y.; Garcia, A.; Hussein, A., Identifying cancer biomarkers by mass spectrometrybased glycomics. Electrophoresis. 2012, 33, (12), 1755-1767. 23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

(19) Tsai, T. H.; Song, E.; Zhu, R.; Di Poto, C.; Wang, M.; Luo, Y.; Varghese, R. S.; Tadesse, M. G.; Ziada, D. H.; Desai, C. S.; Shetty, K.; Mechref, Y.; Ressom, H. W., LC-MS/MS-based serum proteomics for identification of candidate biomarkers for hepatocellular carcinoma. Proteomics. 2015, 15, (13), 23692381. (20) Zacharias, L. G.; Hartmann, A. K.; Song, E.; Zhao, J.; Zhu, R.; Mirzaei, P.; Mechref, Y., HILIC and ERLIC Enrichment of Glycopeptides Derived from Breast and Brain Cancer Cells. J Proteome Res. 2016, 15, (10), 3624-3634. (21) Tang, Z.; Varghese, R. S.; Bekesova, S.; Loffredo, C. A.; Hamid, M. A.; Kyselova, Z.; Mechref, Y.; Novotny, M. V.; Goldman, R.; Ressom, H. W., Identification of N-glycan serum markers associated with hepatocellular carcinoma from mass spectrometry data. J Proteome Res. 2010, 9, (1), 104-112. (22) Mechref, Y.; Hu, Y.; Desantos-Garcia, J. L.; Hussein, A.; Tang, H., Quantitative glycomics strategies. Mol Cell Proteomics. 2013, 12, (4), 874-884. (23) Packer, N. H.; Lawson, M. A.; Jardine, D. R.; Redmond, J. W., A general approach to desalting oligosaccharides released from glycoproteins. Glycoconj J. 1998, 15, (8), 737-747. (24) Hirabayashi, J., Lectin-based structural glycomics: glycoproteomics and glycan profiling. Glycoconj J. 2004, 21, (1-2), 35-40. (25) Zhou, S.; Huang, Y.; Dong, X.; Peng, W.; Veillon, L.; Kitagawa, D. A. S.; Aquino, A. J. A.; Mechref, Y., Isomeric Separation of Permethylated Glycans by Porous Graphitic Carbon (PGC)-LC-MS/MS at High Temperatures. Anal Chem. 2017, 89, (12), 6590-6597. (26) Desantos-Garcia, J. L.; Khalil, S. I.; Hussein, A.; Hu, Y.; Mechref, Y., Enhanced sensitivity of LC-MS analysis of permethylated N-glycans through online purification. Electrophoresis. 2011, 32, (24), 35163525. (27) Madera, M.; Mann, B.; Mechref, Y.; Novotny, M. V., Efficacy of glycoprotein enrichment by microscale lectin affinity chromatography. J Sep Sci. 2008, 31, (14), 2722-2732. (28) Blonder, J.; Goshe, M. B.; Moore, R. J.; Pasa-Tolic, L.; Masselon, C. D.; Lipton, M. S.; Smith, R. D., Enrichment of integral membrane proteins for proteomic analysis using liquid chromatography-tandem mass spectrometry. J Proteome Res. 2002, 1, (4), 351-60. (29) Zielinska, D. F.; Gnad, F.; Wisniewski, J. R.; Mann, M., Precision mapping of an in vivo Nglycoproteome reveals rigid topological and sequence constraints. Cell. 2010, 141, (5), 897-907. (30) Abdul Rahman, S.; Bergstrom, E.; Watson, C. J.; Wilson, K. M.; Ashford, D. A.; Thomas, J. R.; Ungar, D.; Thomas-Oates, J. E., Filter-aided N-glycan separation (FANGS): a convenient sample preparation method for mass spectrometric N-glycan profiling. J Proteome Res. 2014, 13, (3), 1167-1176. (31) Yang, S.; Yuan, W.; Yang, W. M.; Zhou, J. Y.; Harlan, R.; Edwards, J.; Li, S. W.; Zhang, H., Glycan Analysis by Isobaric Aldehyde Reactive Tags and Mass Spectrometry. Anal Chem. 2013, 85, (17), 81888195. (32) Yu, Y. Q.; Gilar, M.; Lee, P. J.; Bouvier, E. S.; Gebler, J. C., Enzyme-friendly, mass spectrometrycompatible surfactant for in-solution enzymatic digestion of proteins. Anal Chem. 2003, 75, (21), 60236028. (33) Zhou, J.; Zhou, T.; Cao, R.; Liu, Z.; Shen, J.; Chen, P.; Wang, X.; Liang, S., Evaluation of the application of sodium deoxycholate to proteomic analysis of rat hippocampal plasma membrane. J Proteome Res. 2006, 5, (10), 2547-2553. (34) Peng, W.; Zhang, Y.; Zhu, R.; Mechref, Y., Comparative Membrane Proteomics Analyses of Breast Cancer Cell Lines to Understand the Molecular Mechanism of Breast Cancer Brain Metastasis. Electrophoresis. 2017, 38, (17), 2124-2134. (35) Masuda, T.; Tomita, M.; Ishihama, Y., Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis. J Proteome Res. 2008, 7, (2), 731-740.

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(36) Leon, I. R.; Schwammle, V.; Jensen, O. N.; Sprenger, R. R., Quantitative assessment of in-solution digestion efficiency identifies optimal protocols for unbiased protein analysis. Mol Cell Proteomics. 2013, 12, (10), 2992-3005. (37) Moore, S. M.; Hess, S. M.; Jorgenson, J. W., Extraction, Enrichment, Solubilization, and Digestion Techniques for Membrane Proteomics. J Proteome Res. 2016, 15, (4), 1243-1252. (38) Kang, P.; Mechref, Y.; Klouckova, I.; Novotny, M. V., Solid-phase permethylation of glycans for mass spectrometric analysis. Rapid Commun Mass Spectrom. 2005, 19, (23), 3421-3428. (39) Mechref, Y.; Kang, P.; Novotny, M. V., Solid-phase permethylation for glycomic analysis. Methods Mol Biol. 2009, 534, 53-64. (40) Dong, X.; Zhou, S.; Mechref, Y., LC-MS/MS analysis of permethylated free oligosaccharides and N-glycans derived from human, bovine, and goat milk samples. Electrophoresis. 2016, 37, (11), 15321548. (41) Huang, Y.; Zhou, S.; Zhu, J.; Lubman, D. M.; Mechref, Y., LC-MS/MS isomeric profiling of permethylated N-glycans derived from serum haptoglobin of hepatocellular carcinoma (HCC) and cirrhotic patients. Electrophoresis. 2017, 38, (17), 2160-2167. (42) Zhou, S.; Dong, X.; Veillon, L.; Huang, Y.; Mechref, Y., LC-MS/MS analysis of permethylated Nglycans facilitating isomeric characterization. Anal Bioanal Chem. 2017, 409, (2), 453-466. (43) Hu, Y. L.; Zhou, S. Y.; Yu, C. Y.; Tang, H. X.; Mechref, Y., Automated annotation and quantitation of glycans by liquid chromatography/electrospray ionization mass spectrometric analysis using the MultiGlycan-ESI computational tool. Rapid Commun Mass Spectrom. 2015, 29, (1), 135-142. (44) Yu, C. Y.; Mayampurath, A.; Hu, Y. L.; Zhou, S. Y.; Mechref, Y.; Tang, H. X., Automated annotation and quantification of glycans using liquid chromatography-mass spectrometry. Bioinformatics. 2013, 29, (13), 1706-1707. (45) Hu, Y.; Shihab, T.; Zhou, S.; Wooding, K.; Mechref, Y., LC-MS/MS of permethylated N-glycans derived from model and human blood serum glycoproteins. Electrophoresis. 2016, 37, (11), 1498-1505. (46) Keller, A.; Nesvizhskii, A. I.; Kolker, E.; Aebersold, R., Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem. 2002, 74, (20), 5383-5392. (47) Nesvizhskii, A. I.; Keller, A.; Kolker, E.; Aebersold, R., A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem. 2003, 75, (17), 4646-4658. (48) Ashburner, M.; Ball, C. A.; Blake, J. A.; Botstein, D.; Butler, H.; Cherry, J. M.; Davis, A. P.; Dolinski, K.; Dwight, S. S.; Eppig, J. T.; Harris, M. A.; Hill, D. P.; Issel-Tarver, L.; Kasarskis, A.; Lewis, S.; Matese, J. C.; Richardson, J. E.; Ringwald, M.; Rubin, G. M.; Sherlock, G., Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000, 25, (1), 25-29. (49) Vizcaino, J. A.; Csordas, A.; del-Toro, N.; Dianes, J. A.; Griss, J.; Lavidas, I.; Mayer, G.; PerezRiverol, Y.; Reisinger, F.; Ternent, T.; Xu, Q. W.; Wang, R.; Hermjakob, H., 2016 update of the PRIDE database and its related tools. Nucleic Acids Res. 2016, 44, (D1), D447-456. (50) Zhou, H.; Froehlich, J. W.; Briscoe, A. C.; Lee, R. S., The GlycoFilter: a simple and comprehensive sample preparation platform for proteomics, N-glycomics and glycosylation site assignment. Mol Cell Proteomics. 2013, 12, (10), 2981-2991. (51) Lin, Y.; Liu, Y.; Li, J.; Zhao, Y.; He, Q.; Han, W.; Chen, P.; Wang, X.; Liang, S., Evaluation and optimization of removal of an acid-insoluble surfactant for shotgun analysis of membrane proteome. Electrophoresis. 2010, 31, (16), 2705-2713. (52) Fu, D.; Chen, L.; O'Neill, R. A., A detailed structural characterization of ribonuclease B oligosaccharides by 1H NMR spectroscopy and mass spectrometry. Carbohydr Res. 1994, 261, (2), 173186.

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(53) ED, G.; G, A.; JU, B.; S, W.; H, V. H., The asparagine-linked oligosaccharides on bovine fetuin. Structural analysis of N-glycanase-released oligosaccharides by 500-megahertz 1H NMR spectroscopy. J Biol Chem. 1988, 263, (34), 18253-18268. (54) Snovida, S. I.; Bodnar, E. D.; Viner, R.; Saba, J.; Perreault, H., A simple cellulose column procedure for selective enrichment of glycopeptides and characterization by nano LC coupled with electron-transfer and high-energy collisional-dissociation tandem mass spectrometry. Carbohydr Res. 2010, 345, (6), 792-801. (55) Selman, M. H.; Hemayatkar, M.; Deelder, A. M.; Wuhrer, M., Cotton HILIC SPE microtips for microscale purification and enrichment of glycans and glycopeptides. Anal Chem. 2011, 83, (7), 24922499. (56) Huang, Y.; Orlando, R., Kinetics of N-Glycan Release from Human Immunoglobulin G (IgG) by PNGase F: All Glycans Are Not Created Equal. J Biomol Tech. 2017, 28, (4), 150-157. (57) Wang, Q.; Christiansen, A.; Samiotakis, A.; Wittung-Stafshede, P.; Cheung, M. S., Comparison of chemical and thermal protein denaturation by combination of computational and experimental approaches. II. J Chem Phys. 2011, 135, (17), 175102. (58) Bennion, B. J.; Daggett, V., The molecular basis for the chemical denaturation of proteins by urea. Proc Natl Acad Sci U S A. 2003, 100, (9), 5142-7.

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Table 1 Table 1. Analytical Merits of the sample preparation protocols evaluated in this study. Sample

Model Glycoprotein mixture (Fetuin + RNase B)

Breast Cancer Cells (MDA-MB-361)

Protocol

Solubilization/Lysis Solution

Cell Lysis

Digestion Solution

Detergent Removal

IS-PBS

10mM PBS

N/A

10mM PBS

N/A

IS-SDC-AP

2.5% SDC+ 10mM PBS

N/A

0.5% SDC+ 10mM PBS

Acidic Precipitation

IS-SDC-PT

2.5% SDC+ 10mM PBS

N/A

0.5% SDC+ 10mM PBS

Phase Transfer

IF-PBS

10mM PBS

N/A

10mM PBS

Filter Aided

IF-SDC

2.5% SDC+ 10mM PBS

N/A

10mM PBS

Filter Aided

IF-SDS

2.5% SDS+ 10mM PBS

N/A

10mM PBS

Filter Aided

PBS-BB

10mM PBS

Beads Beating

10mM PBS

N/A

PBS-F/T

10mM PBS

Freeze/Thaw Cycles

10mM PBS

N/A

PBS-UHS

10mM PBS

Ultrahigh Frequency Sonication

10mM PBS

N/A

SDC-BB

5% SDC+ 10mM PBS

Beads Beating

0.5% SDC+ 10mM PBS

SDC-F/T

5% SDC+ 10mM PBS

Freeze/Thaw Cycles

0.5% SDC+ 10mM PBS

SDC-UHS

5% SDC+ 10mM PBS

Ultrahigh Frequency Sonication

0.5% SDC+ 10mM PBS

Acidic Precipitation Acidic Precipitation Acidic Precipitation

Note: IS: in-solution, IF: in-filter, AP: acid precipitation, PT: phase transfer, BB: beads beating, F/T: freeze/thaw cycles, UHS: ultrahigh sonication.

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Figure legends Figure 1. Experimental design. a. Workflow of model glycoproteins sample preparation; b. Workflow of cell line sample preparation. Figure 2. Quantitative comparison of different protocols using model glycoproteins sample. a. Total intensities of N-glycans derived from RNase B and fetuin by different protocols. N-glycan yields were calculated by summing the peak areas of all RNase B or fetuin glycans. b. Relative distributions of N-glycans derived from RNase B using different protocols. c. Relative distributions of Nglycans derived from fetuin using different protocols. Figure 3. Comparison of total extracted protein and membrane protein identified by LCMS/MS from ~5 million MDA-MB-361 cells using different protocols. a. total extracted protein. b. Venn graph of membrane protein identifications. Beads stands for Beads beating method; F/T stands for freeze/thaw cycling method; UHS stands for ultrahigh frequency sonication method. Figure 4. Qualitative and quantitative comparison of N-glycans released from 1 µg protein (determined by protein assay) extracted from MDA-MB-361 cells using different protocols. a-c. Venn graph of identified N-glycans using different protocols. d-f. Histograms represent the log2(ratio) distributions of all identified N-glycans. This ratio was calculated as the change in abundance for each identified N-glycans in SDC protocol relative to the detergent-free protocols. Figure 5. Relative distributions of N-glycans derived from 50µg proteins extracted from MDA-MB-361 cells using different protocols. a. Relative distributions of high mannose type N-glycans. b. Relative distributions of triantennary type Nglycans.

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Zhu et al. Figure 1

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Zhu et al. Figure 5

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Graphical Abstract: for TOC only

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MS Analysis of N-linked Glycans

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