Sample pretreatment was optimized to obtain high sequence coverage for human serum albumin (HSA, 66.5 kDa) when using nano-electrospray ionization quadrupole time-of-flight mass spectrometry (nESI-Q-TOF-MS). Use of the final method with trypsin, Lys-C and Glu-C digests gave a combined coverage of 98.8%. The addition of peptide fractionation resulted in 99.7% coverage. These results were comparable to those obtained previously with matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The sample pretreatment/nESI-Q-TOF-MS method was also used with collision-induced dissociation to analyze HSA digests and to identify peptides that could be employed as internal mass calibrants in future studies of modifications to HSA

Dodds, Eric D.

Hage, David S.

Kolli, Venkata

Matsuda, Ryan E

Woods, Megan

English

PubMed

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University of Nebraska - Lincoln
DigitalCommons@University of Nebraska - Lincoln
David Hage Publications Published Research - Department of Chemistry
6-2016
Optimizing Sequence Coverage for a Moderate
Mass Protein in Nano-Electrospray Ionization
Quadrupole Time-of-Flight Mass Spectrometry
Ryan E. Matsuda
University of Nebraska-Lincoln, rmatsuda@huskers.unl.edu
Venkata Kolli
University of Nebraska-Lincoln, venkata.kolli@huskers.unl.edu
Megan Woods
University of Nebraska-Lincoln
Eric D. Dodds
University of Nebraska-Lincoln, edodds2@unl.edu
David S. Hage
University of Nebraska - Lincoln, dhage1@unl.edu
Follow this and additional works at: http://digitalcommons.unl.edu/chemistryhage
Part of the Medicinal-Pharmaceutical Chemistry Commons
This Article is brought to you for free and open access by the Published Research - Department of Chemistry at DigitalCommons@University of
Nebraska - Lincoln. It has been accepted for inclusion in David Hage Publications by an authorized administrator of DigitalCommons@University of
Nebraska - Lincoln.
Matsuda, Ryan E.; Kolli, Venkata; Woods, Megan; Dodds, Eric D.; and Hage, David S., "Optimizing Sequence Coverage for a
Moderate Mass Protein in Nano-Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry" (2016). David Hage
Publications. 69.
http://digitalcommons.unl.edu/chemistryhage/69
Accepted Manuscript
Optimizing Sequence Coverage for a Moderate Mass Protein in Nano-Electrospray
Ionization Quadrupole Time-of-Flight Mass Spectrometry
Ryan Matsuda, Venkata Kolli, Megan Woods, Eric D. Dodds, David S. Hage
PII: S0003-2697(16)30141-5
DOI: 10.1016/j.ab.2016.06.014
Reference: YABIO 12416
To appear in: Analytical Biochemistry
Received Date: 21 April 2016
Revised Date: 7 June 2016
Accepted Date: 8 June 2016
Please cite this article as: R. Matsuda, V. Kolli, M. Woods, E.D. Dodds, D.S. Hage, Optimizing
Sequence Coverage for a Moderate Mass Protein in Nano-Electrospray Ionization Quadrupole Time-of-
Flight Mass Spectrometry, Analytical Biochemistry (2016), doi: 10.1016/j.ab.2016.06.014.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.
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Optimizing Sequence Coverage for a Moderate Mass Protein in Nano-Electrospray 
Ionization Quadrupole Time-of-Flight Mass Spectrometry 
 
Ryan Matsuda, Venkata Kolli, Megan Woods, Eric D. Dodds, and David S. Hage* 
 
 
 
Department of Chemistry 
University of Nebraska-Lincoln 
Lincoln, NE 68588-0304 (USA) 
 
 
 
 
*Author for correspondence: 704 Hamilton Hall, Chemistry Department, University of 
Nebraska, Lincoln, NE 68588-0304 USA.  Phone: 402-472-2744; FAX: 402-472-9402; Email: 
dhage1@unl.edu 
  
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Abstract 
Sample pretreatment was optimized to obtain high sequence coverage for human serum 
albumin (HSA, 66.5 kDa) when using nano-electrospray ionization quadrupole time-of-flight 
mass spectrometry (nESI-Q-TOF-MS).  Use of the final method with trypsin, Lys-C and Glu-C 
digests gave a combined coverage of 98.8%.  The addition of peptide fractionation resulted in 
99.7% coverage.  These results were comparable to those obtained previously with matrix-
assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).  The 
sample pretreatment/nESI-Q-TOF-MS method was also used with collision-induced dissociation 
to analyze HSA digests and to identify peptides that could be employed as internal mass 
calibrants in future studies of modifications to HSA.   
 
 
 
 
 
 
 
 
 
 
Keywords: Human serum albumin; Sequence coverage; Sample pretreatment; Nano-
electrospray ionization quadrupole time-of-flight mass spectrometry; Collision-induced 
dissociation; Internal calibration 
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Mass spectrometry (MS) is an important tool for identifying proteins and providing 
information on the primary sequences of proteins and their post-translational modifications 
(PTMs) [1,2].  Several recent studies have used MS to examine human serum albumin (HSA, 
66.5 kDa) and PTMs such as non-enzymatic glycation that occur on this protein [3-5].  The 
effects of such modifications have been of concern given the important role played by HSA in 
transporting many low mass substances in blood (e.g., hormones, fatty acids, and drugs) [6].     
Several past studies have utilized matrix-assisted laser desorption/ionization time-of-
flight mass spectrometry (MALDI-TOF-MS) to investigate the structure of HSA and glycated 
HSA [2,3,7-12].  Sequence coverages of up to 97.4% have been reached by optimizing the 
MALDI matrix and conditions that were used for sample pretreatment; however, obtaining this 
level of coverage required multiple enzyme digestions and a series of steps for peptide 
fractionation [2].  The purpose of this current study was to determine if more convenient 
pretreatment conditions could still obtain high sequence coverage for a moderately high mass 
protein such as HSA and when using nano-electrospray quadrupole time-of-flight mass 
spectrometry (nESI-Q-TOF-MS) for analysis of the digests. 
  Two pretreatment procedures were initially considered (see Supplementary Material for 
more details).  Pretreatment Method 1 followed a scheme similar to the previous method used 
with MALDI-TOF-MS [2] but without any peptide fractionation. In this method, a sample of 
HSA was first treated by means of alkylation and reduction, followed by dialysis to remove any 
excess reagents.  The protein sample was then digested separately with three proteolytic enzymes 
(i.e., trypsin, Lys-C and Glu-C) and analyzed by MS.  In Pretreatment Method 2, all of the 
pretreatment and digestion processes were performed sequentially in a single vial, resulting in 
the ability to use lower concentrations of both the protein and pretreatment reagents.  The 
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pretreatment conditions for alkylation and reduction were similar to those in Pretreatment 
Method 1, with some slight variations in the reaction times and temperatures. Also in 
Pretreatment Method 2, dialysis was no longer used to remove the excess pretreatment reagents, 
and each digest was fractionated by using ZipTipµ-C18 pipette tips. 
Figure 1 and the Supplementary Material summarize the results that were obtained by 
nESI-Q-TOF-MS for digests of HSA when using these pretreatment methods.  The use of 
Pretreatment Method 1 with a tryptic digest gave 42 identified peptides and a sequence coverage 
of 36.9% for HSA, while the Glu-C or Lys-C digests gave sequence coverages of 39.1% or 
32.1% and 43 or 21 identified peptides, respectively.  For Pretreatment Method 2, the sequence 
coverages were 86.0% for the tryptic digest, 84.1% for Lys-C digest and 78.2% for Glu-C digest, 
which corresponded to 104, 52, or 54 identified peptides in these digests.  Because there was 
both improved sequence coverage and an increase in the number of identified peptides with 
Pretreatment Method 2, this method was selected for use in the remainder of this study.  
The results obtained by Pretreatment Method 2, nESI-Q-TOF-MS, and using a single 
elution step for peptide fractionation were compared to the results from the previous 
pretreatment method that had been used with MALDI-TOF-MS to examine HSA (see 
Supplementary Material) [2].  The previous MALDI-TOF-MS method gave sequence coverages 
of 82.9%, 77.6% or 64.6% and 56, 41, or 28 identified peptides when HSA was digested with 
trypsin, Lys-C or Glu-C, respectively, and these digests were fractionated on ZipTipµ-C18 tips 
through a series of several elution steps [2].  The approach based on Pretreatment Method 2 and 
nESI-Q-TOF-MS, which used a simpler alkylation/reduction protocol and only a single elution 
step for peptide fractionation, gave 3.1-13.6% higher sequence coverages for these digests and a 
greater number of identifiable peaks (i.e., 11-48 more peptides per digest). The use of multiple 
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elution steps for peptide fractionation, as described in the Supplementary Material, gave a small 
increase in the sequence coverages for Pretreatment Method 2 and nESI-Q-TOF-MS to levels of 
83.2% and 91.5% for tryptic and Glu-C digests and a slight decrease to 72.6% for the Lys-C 
digest.    
When a single elution step was used for peptide fractionation in Pretreatment Method 2, 
the total sequence coverage obtained by nESI-Q-TOF-MS for HSA was 98.8%.  This coverage 
included 578 out of the 585 amino acids in HSA, with the only excluded region being residues 
535-541.  This coverage included 56 out of 59 lysines in HSA and all of the arginine residues, 
which were of interest because lysine and arginine are both potential sites for glycation-related 
modifications (Note: the three lysines in residues 535-541 that were not included in this coverage 
are not major sites for such modifications) [3].  When multiple elution steps were used for 
peptide fractionation, the combined digests gave an overall sequence coverage was 99.7%, in 
which a total of 583 out of 585 residues were now covered.  The only missing residues in this 
second case came from residues 312-313; however, previous reports have shown that K313 is 
highly susceptible to modification by glycation [3].   
Both of these situations were an improvement over the previous pretreatment method 
used with MALDI-TOF-MS, in which a total sequence coverage of 97.4% was obtained for HSA 
when data were combined for the three enzyme digests [2].  In this prior work, 570 out of the 
585 amino acids were included, as well as 54 of the 59 lysines and all of the arginines.  In the 
missed regions for this previous method, K4, K536, K538, and K541 are not major sites for 
glycation-related modifications, but K317 can be an important location for such PTMs [3]. 
Neither the previous MALDI-TOF-MS method nor the use of Pretreatment Method 2 with nESI-
Q-TOF-MS and a single elution step for fractionation covered the region 535-541 of HSA. 
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However, if the data were combined for nESI-Q-TOF-MS and Pretreatment Method 2 when 
using both a single step and multiple steps for peptide fractionation, then all 585 amino acids of 
HSA included and effectively 100% coverage was achieved.  
In the previous work with HSA and MALDI-TOF-MS [2], 0.5 µL portions of the 
fractionated protein digests (protein concentrations: ~0.822 µg/µL in tryptic digest, ~0.870 
µg/µL in Lys-C digest, and ~ 1.25 µg/µL in Glu-C digest) were individually mixed with the 
MALDI matrix and spotted onto a plate for MS analysis.  These multiple fractions increased the 
overall amount of sample that was needed for analysis.  Although a 20 µL sample of the purified 
protein digest (protein concentration: ~ 1 µg/µL in each digest) was prepared for use in 
Pretreatment Method 2 and nESI-Q-TOF-MS, the sample could be placed directly into the nano-
ESI emitter and sprayed in nanoliter volumes to obtain a mass spectrum.  In addition, the 
remaining sample from the nano-ESI emitter could be recovered and stored for future 
experiments.   
Another advantage of the nESI-Q-TOF-MS method over MALDI-TOF-MS was its 
ability to perform MS/MS experiments on the digests, such as by using CID to create peptide 
fragmentation [13]. This type of experiment was performed on the various digests of HSA to 
confirm the identity of the peptides, as demonstrated in Figure 2(a).  Some of these peptides were 
also tested for use as internal m/z calibrants to improve mass accuracy when searching for PTMs 
[14,15] like glycation-related modifications [2,3,7-10], as illustrated in Figure 2(b).  Around 6-7 
unique peptides were selected through these experiments from each type of enzyme digest of 
HSA, with each peptide having a relatively high signal intensity (see Supplementary Material).  
One of these peptides is shown in Figure 2(a) and corresponded to residues 337-348 and 
RHPDYSVVLLLR; this peptide was selected because it contained no lysine residues and would 
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not be subject to PTMs at such sites.  Other peptides were chosen because they appeared in a 
variety of regions in the mass spectra.  
These peptides were next used as internal calibrants in the analysis of a separate set of 
HSA digests that were prepared by using trypsin, Lys-C, or Glu-C.  A calibrated spectrum of a 
Glu-C digest of HSA resulted in a root mean square mass error of 19.9 ppm, with similar results 
being obtained with the trypsin and Lys-C digests.  This low mass measurement error made it 
possible to lower the mass tolerance that was used for sequence analysis (i.e., a previous value of 
50 ppm); this change had the advantage of reducing the possibility of false positive matches 
while also improving confidence in the assignment of putative peptides, including those 
harboring PTMs. The effect of this change on the sequence coverage was evaluated by using the 
internally calibrated mass spectra for the tryptic, Lys-C and Glu-C digests along with a 25 ppm 
mass tolerance.  Under these conditions, the individual digests had sequence coverages of 79.8%, 
86.5%, and 78.9% for tryptic, Lys-C and Glu-C digests, with a 96.1% total coverage for the 
combined results.  These results were comparable to the previous values acquired by MALDI-
TOF-MS [2] and were only slightly lower than the coverages seen earlier with nESI-Q-TOF-MS 
when using a 50 ppm mass tolerance and no internal calibration.    
 In summary, a sample pretreatment was developed for use with nESI-Q-TOF-MS to 
obtain high sequence coverage for HSA.  The combined use of nESI-Q-TOF-MS and the final 
pretreatment method with single or multiple elution steps for peptide fractionation gave sequence 
coverages of 98.8-99.7% for HSA, with a combined coverage of essentially 100%.  It was also 
shown how this combined approach could be used with CID to confirm the structures of the 
peptides and to select some of these peptides for use as internal calibrants in MS/MS 
experiments.  Although this study focused on the analysis of HSA by nESI-Q-TOF-MS, the same 
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methods could be applied to other proteins with moderately high masses. 
 
Acknowledgements 
This work was funded by the NIH under grant R01 DK069629.  R. Matsuda was 
supported under a fellowship through the Molecular Mechanisms of Disease program at the 
University of Nebraska. 
 
References 
1. R. Aebersold, M. Mann, Mass spectrometry-based proteomics, Nature 422 (2003) 198-
207. 
2. C. Wa, R. Cerny, D.S. Hage, Obtaining high sequence coverage in matrix-assisted laser 
desorption time-of-flight mass spectrometry for studies of protein modification: analysis 
of human serum albumin as a model. Anal. Biochem. 349 (2006) 229-241. 
3. J. Anguizola, R. Matsuda, O.S. Barnaby, K.S. Joseph, C. Wa, E. Debolt, M. Koke, D.S. 
Hage, Review: glycation of human serum albumin. Clin. Chim. Acta 425 (2013) 64–76. 
4. A. Lapolla, D. Fedele, R. Seraglia, P. Traldi, The role of mass spectrometry in the study 
of non-enzymatic protein glycation in diabetes: an update, Mass Spectrom. Rev. 25 
(2006) 775-797. 
5. Q. Zhang, J.M. Ames, R.D. Smith, J.W. Baynes, T.O. Metz, A perspective on the 
Maillard reaction and the analysis of protein glycation by mass spectrometry: probing the 
pathogenesis of chronic disease, J. Proteome Res. 8 (2009) 754-769. 
6. T. Peters, Jr., All About Albumin: Biochemistry, Genetics, and Medical Applications. 
Academic Press, San Diego, 1996. 
7. O.S. Barnaby, R.L. Cerny, W. Clarke, D.S. Hage, Comparison of modification sites 
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formed on human serum albumin at various stages of glycation. Clin. Chim. Acta 412 
(2011) 277-285. 
8. O.S. Barnaby, R.L. Cerny, W. Clarke, D.S. Hage, Quantitative analysis of glycation 
patterns in human serum albumin using 16O/18O-labeling and MALDI-TOF MS. Clin. 
Chim. Acta 412 (2011) 1606-1615. 
9. O.S. Barnaby, C. Wa, R.L. Cerny, W. Clarke, D.S. Hage, Quantitative analysis of 
glycation sites on human serum albumin using 16O/18O-labeling and matrix-assisted laser 
desorption/ionization time-of-flight mass spectrometry, Clin. Chim. Acta 411 (2010) 
1102-1110. 
10. C. Wa, R. Cerny, W. Clarke, D.S. Hage, Characterization of glycation adducts on human 
serum albumin by matrix-assisted laser desorption ionization time-of-flight mass 
spectrometry, Clin. Chim. Acta 385 (2007) 48-60. 
11. F.L. Brancia, J.Z. Bereszczak, A. Lapolla, D. Fedele, L. Baccarin, R. Seraglia, P. Traldi, 
Comprehensive analysis of glycated human serum albumin tryptic peptides by off-line 
liquid chromatography followed by MALDI analysis on a time-of-flight/curved field 
reflectron tandem mass spectrometer, J. Mass Spectrom. 41 (2006) 1179–1185. 
12. B.S. Lee, S. Krishnanchettiar, S.S. Lateef, S. Gupta, Analyses of the in vitro 
nonenzymatic glycation of peptides/proteins by matrix-assisted laser 
desorption/ionization mass spectrometry, Int. J. Mass Spectrom. 260 (2007) 67–74. 
13. J.H. Gross, Mass Spectrometry, A Textbook, 2nd ed., Springer, New York, 2004. 
14. K. Hjerno, P Hojrup, in R. Mattiesen (Ed.), Mass Spectrometry Data Analysis in 
Proteomics, Humana Press, Totowa, 2007, Chap. 3. 
15. B. Xiang, M. Prado, An accurate and clean calibration method for MALDI-MS, J. 
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Biomol. Tech. 21 (2010) 116-119. 
 
  
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Figure Legends 
Figure 1. Primary sequence of human serum albumin (HSA) [2] and total sequence 
coverage of HSA that was obtained with Pretreatment Method 2 and nESI-Q-
TOF-MS when combining the sequence coverages for tryptic (shown in 
underline), Lys-C (in bold), and Glu-C (in italics) digests.  These results were 
obtained using a tolerance level that was equal to a maximum 50.0 ppm mass 
difference for each positive match. 
 
Figure 2. (a) MS/MS spectrum for the fragmentation of a peptide with a mass-to-charge 
ratio of 734 m/z; this peptide corresponded to the 373-348 region of HSA, which 
has a sequence of RHPDYSVVLLLR. (b) Mass spectrum of a trypsin-digested 
HSA sample, in which peaks that were selected for internal calibration are 
indicated by an asterisk (*).   
 
 
 
 
 
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Residue No. Sequence
1-40 DAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHV
41-80 KLVNEVTEFA KTCVADESAE NCDKSLHTLF GDKLCTVATL
81-120 RETYGEMADC CAKQEPERNE CFLQHKDDNP NLPRLVRPEV
121-160 DVMCTAFHDN EETFLKKYLY EIARRHPYFY APELLFFAKR
161-200 YKAAFTECCQ AADKAACLLP KLDELRDEGK ASSAKQRLKC
201-240 ASLQKFGERA FKAWAVARLS QRFPKAEFAE VSKLVTDLTK
241-280 VHTECCHGDL LECADDRADL AKYICENQDS ISSKLKECCE
281-320 KPLLEKSHCI AEVENDEMPA DLPSLAADFV ESKDVCKNYA
321-360 EAKDVFLGMF LYEYARRHPD YSVVLLLRLA KTYETTLEKC
361-400 CAAADPHECY AKVFDEFKPL VEEPQNLIKQ NCELFEQLGE
401-440 YKFQNALLVR YTKKVPQVST PTLVEVSRNL GKVGSKCCKH
441-480 PEAKRMPCAE DYLSVVLNQL CVLHEKTPVS DRVTKCCTES
481-520 LVNRRPCFSA LEVDETYVPK EFNAETFTFH ADICTLSEKE
521-560 RQIKKQTALV ELVKHKPKAT KEQLKAVMDD FAAFVEKCCK
561-585 ADDKETCFAE EGKKLVASSQ AALGL
Figure 1
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Mass (m/z)
Mass (m/z)
R
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In
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R
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In
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(b)
Figure 2


Optimizing Sequence Coverage for a Moderate Mass Protein in Nano-Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry

DigitalCommons@University of Nebraska

University of Nebraska - LincolnDigitalCommons@University of Nebraska - LincolnDavid Hage Publications Published Research - Department of Chemistry6-2016Optimizing Sequence Coverage for a ModerateMass Protein in Nano-Electrospray IonizationQuadrupole Time-of-Flight Mass SpectrometryRyan E. MatsudaUniversity of Nebraska-Lincoln, rmatsuda@huskers.unl.eduVenkata KolliUniversity of Nebraska-Lincoln, venkata.kolli@huskers.unl.eduMegan WoodsUniversity of Nebraska-LincolnEric D. DoddsUniversity of Nebraska-Lincoln, edodds2@unl.eduDavid S. HageUniversity of Nebraska - Lincoln, dhage1@unl.eduFollow this and additional works at: http://digitalcommons.unl.edu/chemistryhagePart of the Medicinal-Pharmaceutical Chemistry CommonsThis Article is brought to you for free and open access by the Published Research - Department of Chemistry at DigitalCommons@University ofNebraska - Lincoln. It has been accepted for inclusion in David Hage Publications by an authorized administrator of DigitalCommons@University ofNebraska - Lincoln.Matsuda, Ryan E.; Kolli, Venkata; Woods, Megan; Dodds, Eric D.; and Hage, David S., "Optimizing Sequence Coverage for aModerate Mass Protein in Nano-Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry" (2016). David HagePublications. 69.http://digitalcommons.unl.edu/chemistryhage/69Accepted ManuscriptOptimizing Sequence Coverage for a Moderate Mass Protein in Nano-ElectrosprayIonization Quadrupole Time-of-Flight Mass SpectrometryRyan Matsuda, Venkata Kolli, Megan Woods, Eric D. Dodds, David S. HagePII: S0003-2697(16)30141-5DOI: 10.1016/j.ab.2016.06.014Reference: YABIO 12416To appear in: Analytical BiochemistryReceived Date: 21 April 2016Revised Date: 7 June 2016Accepted Date: 8 June 2016Please cite this article as: R. Matsuda, V. Kolli, M. Woods, E.D. Dodds, D.S. Hage, OptimizingSequence Coverage for a Moderate Mass Protein in Nano-Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry, Analytical Biochemistry (2016), doi: 10.1016/j.ab.2016.06.014.This is a PDF file of an unedited manuscript that has been accepted for publication. As a service toour customers we are providing this early version of the manuscript. The manuscript will undergocopyediting, typesetting, and review of the resulting proof before it is published in its final form. Pleasenote that during the production process errors may be discovered which could affect the content, and alllegal disclaimers that apply to the journal pertain.MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT1     Optimizing Sequence Coverage for a Moderate Mass Protein in Nano-Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry  Ryan Matsuda, Venkata Kolli, Megan Woods, Eric D. Dodds, and David S. Hage*    Department of Chemistry University of Nebraska-Lincoln Lincoln, NE 68588-0304 (USA)     *Author for correspondence: 704 Hamilton Hall, Chemistry Department, University of Nebraska, Lincoln, NE 68588-0304 USA.  Phone: 402-472-2744; FAX: 402-472-9402; Email: dhage1@unl.edu   MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT2  Abstract Sample pretreatment was optimized to obtain high sequence coverage for human serum albumin (HSA, 66.5 kDa) when using nano-electrospray ionization quadrupole time-of-flight mass spectrometry (nESI-Q-TOF-MS).  Use of the final method with trypsin, Lys-C and Glu-C digests gave a combined coverage of 98.8%.  The addition of peptide fractionation resulted in 99.7% coverage.  These results were comparable to those obtained previously with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).  The sample pretreatment/nESI-Q-TOF-MS method was also used with collision-induced dissociation to analyze HSA digests and to identify peptides that could be employed as internal mass calibrants in future studies of modifications to HSA.             Keywords: Human serum albumin; Sequence coverage; Sample pretreatment; Nano-electrospray ionization quadrupole time-of-flight mass spectrometry; Collision-induced dissociation; Internal calibration MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT3  Mass spectrometry (MS) is an important tool for identifying proteins and providing information on the primary sequences of proteins and their post-translational modifications (PTMs) [1,2].  Several recent studies have used MS to examine human serum albumin (HSA, 66.5 kDa) and PTMs such as non-enzymatic glycation that occur on this protein [3-5].  The effects of such modifications have been of concern given the important role played by HSA in transporting many low mass substances in blood (e.g., hormones, fatty acids, and drugs) [6].     Several past studies have utilized matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to investigate the structure of HSA and glycated HSA [2,3,7-12].  Sequence coverages of up to 97.4% have been reached by optimizing the MALDI matrix and conditions that were used for sample pretreatment; however, obtaining this level of coverage required multiple enzyme digestions and a series of steps for peptide fractionation [2].  The purpose of this current study was to determine if more convenient pretreatment conditions could still obtain high sequence coverage for a moderately high mass protein such as HSA and when using nano-electrospray quadrupole time-of-flight mass spectrometry (nESI-Q-TOF-MS) for analysis of the digests.   Two pretreatment procedures were initially considered (see Supplementary Material for more details).  Pretreatment Method 1 followed a scheme similar to the previous method used with MALDI-TOF-MS [2] but without any peptide fractionation. In this method, a sample of HSA was first treated by means of alkylation and reduction, followed by dialysis to remove any excess reagents.  The protein sample was then digested separately with three proteolytic enzymes (i.e., trypsin, Lys-C and Glu-C) and analyzed by MS.  In Pretreatment Method 2, all of the pretreatment and digestion processes were performed sequentially in a single vial, resulting in the ability to use lower concentrations of both the protein and pretreatment reagents.  The MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT4  pretreatment conditions for alkylation and reduction were similar to those in Pretreatment Method 1, with some slight variations in the reaction times and temperatures. Also in Pretreatment Method 2, dialysis was no longer used to remove the excess pretreatment reagents, and each digest was fractionated by using ZipTipµ-C18 pipette tips. Figure 1 and the Supplementary Material summarize the results that were obtained by nESI-Q-TOF-MS for digests of HSA when using these pretreatment methods.  The use of Pretreatment Method 1 with a tryptic digest gave 42 identified peptides and a sequence coverage of 36.9% for HSA, while the Glu-C or Lys-C digests gave sequence coverages of 39.1% or 32.1% and 43 or 21 identified peptides, respectively.  For Pretreatment Method 2, the sequence coverages were 86.0% for the tryptic digest, 84.1% for Lys-C digest and 78.2% for Glu-C digest, which corresponded to 104, 52, or 54 identified peptides in these digests.  Because there was both improved sequence coverage and an increase in the number of identified peptides with Pretreatment Method 2, this method was selected for use in the remainder of this study.  The results obtained by Pretreatment Method 2, nESI-Q-TOF-MS, and using a single elution step for peptide fractionation were compared to the results from the previous pretreatment method that had been used with MALDI-TOF-MS to examine HSA (see Supplementary Material) [2].  The previous MALDI-TOF-MS method gave sequence coverages of 82.9%, 77.6% or 64.6% and 56, 41, or 28 identified peptides when HSA was digested with trypsin, Lys-C or Glu-C, respectively, and these digests were fractionated on ZipTipµ-C18 tips through a series of several elution steps [2].  The approach based on Pretreatment Method 2 and nESI-Q-TOF-MS, which used a simpler alkylation/reduction protocol and only a single elution step for peptide fractionation, gave 3.1-13.6% higher sequence coverages for these digests and a greater number of identifiable peaks (i.e., 11-48 more peptides per digest). The use of multiple MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT5  elution steps for peptide fractionation, as described in the Supplementary Material, gave a small increase in the sequence coverages for Pretreatment Method 2 and nESI-Q-TOF-MS to levels of 83.2% and 91.5% for tryptic and Glu-C digests and a slight decrease to 72.6% for the Lys-C digest.    When a single elution step was used for peptide fractionation in Pretreatment Method 2, the total sequence coverage obtained by nESI-Q-TOF-MS for HSA was 98.8%.  This coverage included 578 out of the 585 amino acids in HSA, with the only excluded region being residues 535-541.  This coverage included 56 out of 59 lysines in HSA and all of the arginine residues, which were of interest because lysine and arginine are both potential sites for glycation-related modifications (Note: the three lysines in residues 535-541 that were not included in this coverage are not major sites for such modifications) [3].  When multiple elution steps were used for peptide fractionation, the combined digests gave an overall sequence coverage was 99.7%, in which a total of 583 out of 585 residues were now covered.  The only missing residues in this second case came from residues 312-313; however, previous reports have shown that K313 is highly susceptible to modification by glycation [3].   Both of these situations were an improvement over the previous pretreatment method used with MALDI-TOF-MS, in which a total sequence coverage of 97.4% was obtained for HSA when data were combined for the three enzyme digests [2].  In this prior work, 570 out of the 585 amino acids were included, as well as 54 of the 59 lysines and all of the arginines.  In the missed regions for this previous method, K4, K536, K538, and K541 are not major sites for glycation-related modifications, but K317 can be an important location for such PTMs [3]. Neither the previous MALDI-TOF-MS method nor the use of Pretreatment Method 2 with nESI-Q-TOF-MS and a single elution step for fractionation covered the region 535-541 of HSA. MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT6  However, if the data were combined for nESI-Q-TOF-MS and Pretreatment Method 2 when using both a single step and multiple steps for peptide fractionation, then all 585 amino acids of HSA included and effectively 100% coverage was achieved.  In the previous work with HSA and MALDI-TOF-MS [2], 0.5 µL portions of the fractionated protein digests (protein concentrations: ~0.822 µg/µL in tryptic digest, ~0.870 µg/µL in Lys-C digest, and ~ 1.25 µg/µL in Glu-C digest) were individually mixed with the MALDI matrix and spotted onto a plate for MS analysis.  These multiple fractions increased the overall amount of sample that was needed for analysis.  Although a 20 µL sample of the purified protein digest (protein concentration: ~ 1 µg/µL in each digest) was prepared for use in Pretreatment Method 2 and nESI-Q-TOF-MS, the sample could be placed directly into the nano-ESI emitter and sprayed in nanoliter volumes to obtain a mass spectrum.  In addition, the remaining sample from the nano-ESI emitter could be recovered and stored for future experiments.   Another advantage of the nESI-Q-TOF-MS method over MALDI-TOF-MS was its ability to perform MS/MS experiments on the digests, such as by using CID to create peptide fragmentation [13]. This type of experiment was performed on the various digests of HSA to confirm the identity of the peptides, as demonstrated in Figure 2(a).  Some of these peptides were also tested for use as internal m/z calibrants to improve mass accuracy when searching for PTMs [14,15] like glycation-related modifications [2,3,7-10], as illustrated in Figure 2(b).  Around 6-7 unique peptides were selected through these experiments from each type of enzyme digest of HSA, with each peptide having a relatively high signal intensity (see Supplementary Material).  One of these peptides is shown in Figure 2(a) and corresponded to residues 337-348 and RHPDYSVVLLLR; this peptide was selected because it contained no lysine residues and would MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT7  not be subject to PTMs at such sites.  Other peptides were chosen because they appeared in a variety of regions in the mass spectra.  These peptides were next used as internal calibrants in the analysis of a separate set of HSA digests that were prepared by using trypsin, Lys-C, or Glu-C.  A calibrated spectrum of a Glu-C digest of HSA resulted in a root mean square mass error of 19.9 ppm, with similar results being obtained with the trypsin and Lys-C digests.  This low mass measurement error made it possible to lower the mass tolerance that was used for sequence analysis (i.e., a previous value of 50 ppm); this change had the advantage of reducing the possibility of false positive matches while also improving confidence in the assignment of putative peptides, including those harboring PTMs. The effect of this change on the sequence coverage was evaluated by using the internally calibrated mass spectra for the tryptic, Lys-C and Glu-C digests along with a 25 ppm mass tolerance.  Under these conditions, the individual digests had sequence coverages of 79.8%, 86.5%, and 78.9% for tryptic, Lys-C and Glu-C digests, with a 96.1% total coverage for the combined results.  These results were comparable to the previous values acquired by MALDI-TOF-MS [2] and were only slightly lower than the coverages seen earlier with nESI-Q-TOF-MS when using a 50 ppm mass tolerance and no internal calibration.     In summary, a sample pretreatment was developed for use with nESI-Q-TOF-MS to obtain high sequence coverage for HSA.  The combined use of nESI-Q-TOF-MS and the final pretreatment method with single or multiple elution steps for peptide fractionation gave sequence coverages of 98.8-99.7% for HSA, with a combined coverage of essentially 100%.  It was also shown how this combined approach could be used with CID to confirm the structures of the peptides and to select some of these peptides for use as internal calibrants in MS/MS experiments.  Although this study focused on the analysis of HSA by nESI-Q-TOF-MS, the same MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT8  methods could be applied to other proteins with moderately high masses.  Acknowledgements This work was funded by the NIH under grant R01 DK069629.  R. Matsuda was supported under a fellowship through the Molecular Mechanisms of Disease program at the University of Nebraska.  References 1. R. Aebersold, M. Mann, Mass spectrometry-based proteomics, Nature 422 (2003) 198-207. 2. C. Wa, R. Cerny, D.S. Hage, Obtaining high sequence coverage in matrix-assisted laser desorption time-of-flight mass spectrometry for studies of protein modification: analysis of human serum albumin as a model. Anal. Biochem. 349 (2006) 229-241. 3. J. Anguizola, R. Matsuda, O.S. Barnaby, K.S. Joseph, C. Wa, E. Debolt, M. Koke, D.S. Hage, Review: glycation of human serum albumin. Clin. Chim. Acta 425 (2013) 64–76. 4. A. Lapolla, D. Fedele, R. Seraglia, P. Traldi, The role of mass spectrometry in the study of non-enzymatic protein glycation in diabetes: an update, Mass Spectrom. Rev. 25 (2006) 775-797. 5. Q. Zhang, J.M. Ames, R.D. Smith, J.W. Baynes, T.O. Metz, A perspective on the Maillard reaction and the analysis of protein glycation by mass spectrometry: probing the pathogenesis of chronic disease, J. Proteome Res. 8 (2009) 754-769. 6. T. Peters, Jr., All About Albumin: Biochemistry, Genetics, and Medical Applications. Academic Press, San Diego, 1996. 7. O.S. Barnaby, R.L. Cerny, W. Clarke, D.S. Hage, Comparison of modification sites MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT9  formed on human serum albumin at various stages of glycation. Clin. Chim. Acta 412 (2011) 277-285. 8. O.S. Barnaby, R.L. Cerny, W. Clarke, D.S. Hage, Quantitative analysis of glycation patterns in human serum albumin using 16O/18O-labeling and MALDI-TOF MS. Clin. Chim. Acta 412 (2011) 1606-1615. 9. O.S. Barnaby, C. Wa, R.L. Cerny, W. Clarke, D.S. Hage, Quantitative analysis of glycation sites on human serum albumin using 16O/18O-labeling and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Clin. Chim. Acta 411 (2010) 1102-1110. 10. C. Wa, R. Cerny, W. Clarke, D.S. Hage, Characterization of glycation adducts on human serum albumin by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, Clin. Chim. Acta 385 (2007) 48-60. 11. F.L. Brancia, J.Z. Bereszczak, A. Lapolla, D. Fedele, L. Baccarin, R. Seraglia, P. Traldi, Comprehensive analysis of glycated human serum albumin tryptic peptides by off-line liquid chromatography followed by MALDI analysis on a time-of-flight/curved field reflectron tandem mass spectrometer, J. Mass Spectrom. 41 (2006) 1179–1185. 12. B.S. Lee, S. Krishnanchettiar, S.S. Lateef, S. Gupta, Analyses of the in vitro nonenzymatic glycation of peptides/proteins by matrix-assisted laser desorption/ionization mass spectrometry, Int. J. Mass Spectrom. 260 (2007) 67–74. 13. J.H. Gross, Mass Spectrometry, A Textbook, 2nd ed., Springer, New York, 2004. 14. K. Hjerno, P Hojrup, in R. Mattiesen (Ed.), Mass Spectrometry Data Analysis in Proteomics, Humana Press, Totowa, 2007, Chap. 3. 15. B. Xiang, M. Prado, An accurate and clean calibration method for MALDI-MS, J. MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT10  Biomol. Tech. 21 (2010) 116-119.    MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPT11  Figure Legends Figure 1. Primary sequence of human serum albumin (HSA) [2] and total sequence coverage of HSA that was obtained with Pretreatment Method 2 and nESI-Q-TOF-MS when combining the sequence coverages for tryptic (shown in underline), Lys-C (in bold), and Glu-C (in italics) digests.  These results were obtained using a tolerance level that was equal to a maximum 50.0 ppm mass difference for each positive match.  Figure 2. (a) MS/MS spectrum for the fragmentation of a peptide with a mass-to-charge ratio of 734 m/z; this peptide corresponded to the 373-348 region of HSA, which has a sequence of RHPDYSVVLLLR. (b) Mass spectrum of a trypsin-digested HSA sample, in which peaks that were selected for internal calibration are indicated by an asterisk (*).        MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPTResidue No. Sequence1-40 DAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHV41-80 KLVNEVTEFA KTCVADESAE NCDKSLHTLF GDKLCTVATL81-120 RETYGEMADC CAKQEPERNE CFLQHKDDNP NLPRLVRPEV121-160 DVMCTAFHDN EETFLKKYLY EIARRHPYFY APELLFFAKR161-200 YKAAFTECCQ AADKAACLLP KLDELRDEGK ASSAKQRLKC201-240 ASLQKFGERA FKAWAVARLS QRFPKAEFAE VSKLVTDLTK241-280 VHTECCHGDL LECADDRADL AKYICENQDS ISSKLKECCE281-320 KPLLEKSHCI AEVENDEMPA DLPSLAADFV ESKDVCKNYA321-360 EAKDVFLGMF LYEYARRHPD YSVVLLLRLA KTYETTLEKC361-400 CAAADPHECY AKVFDEFKPL VEEPQNLIKQ NCELFEQLGE401-440 YKFQNALLVR YTKKVPQVST PTLVEVSRNL GKVGSKCCKH441-480 PEAKRMPCAE DYLSVVLNQL CVLHEKTPVS DRVTKCCTES481-520 LVNRRPCFSA LEVDETYVPK EFNAETFTFH ADICTLSEKE521-560 RQIKKQTALV ELVKHKPKAT KEQLKAVMDD FAAFVEKCCK561-585 ADDKETCFAE EGKKLVASSQ AALGLFigure 1MANUSCRIPT ACCEPTEDACCEPTED MANUSCRIPTMass (m/z)Mass (m/z)Relative IntensityRelative Intensity(a)(b)Figure 2

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Optimizing Sequence Coverage for a Moderate Mass Protein in Nano-Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry

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