For Peer Review - Digital CSICdigital.csic.es/bitstream/10261/150457/4/iron-binding... · 2018-09-19 · For Peer Review 1 1 INTERPRETIVE SUMMARY 2 Short communication: Identification

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INTERPRETIVE SUMMARY 1

Short communication: Identification of iron-binding peptides from whey protein 2

hydrolyzates using IMAC-Fe (III) and RP-HPLC-MS/MS. By Cruz-Huerta et al. For the 3

first time, the identification of peptide sequences with iron-binding capacity from whey 4

proteins has been performed. Better knowledge of the relationship between peptide structure 5

and iron-chelating activity has been provided. The enrichment of iron-chelating amino acids 6

(Asp, Glu and Pro) and the existence of favoured protein domains have been established. This 7

study endorses the promising role of whey protein hydrolyzates as functional ingredients in 8

iron supplementation treatments. 9

10

RUNNING HEAD. SHORT COMMUNICATION: IDENTIFICATION OF WHEY IRON-11

BINDING PEPTIDES 12

13

Short communication: Identification of iron-binding peptides from whey protein 14

hydrolyzates using IMAC-Fe (III) and RP-HPLC-MS/MS 15

16

Elvia Cruz-Huerta*, Daniel Martínez-Maqueda

*, Isidra Recio

*, Lucia de la Hoz

†, Vera S. 17

Nunes da Silva†, Maria Teresa Bertoldo Pacheco

†, Lourdes Amigo

*1 18

19

*Instituto de Investigación en Ciencias de la Alimentación, CIAL (CSIC-UAM, CEI 20

UAM+CSIC), Nicolás Cabrera, 9, 28049 Madrid, Spain 21

2Instituto de Tecnología de Alimentos (CCQA, ITAL), Av. Brasil, 2880, CP 139, CEP 13070-22

178, Campinas, SP, Brazil

23

24

1 Corresponding author: Dr. Lourdes Amigo Garrido 25

Phone: +34 910017939 26

Fax: +3491001905 27

E-mail address: [email protected] 28

29

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Journal of Dairy Science

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ABSTRACT 30

The peptides with iron-binding capacity obtained by hydrolysis of whey protein with 31

Alcalase, pancreatin and Flavourzyme were identified. Hydrolyzates were subjected to 32

IMAC-Fe (III) chromatography and the bound peptides were sequenced by mass 33

spectrometry. Regardless the enzyme used, the domains f(42-59) and f(125-137) from β-34

lactoglobulin enclosed most of identified peptides. This trend was less pronounced in the case 35

of peptides derived from α-lactalbumin, with sequences from diverse regions. Iron-bound 36

peptides exhibited common structural characteristics, such as, the abundance of Asp, Glu and 37

Pro, as revealed by mass spectrometry and amino acid analysis. In conclusion, the 38

characterization of iron-binding peptides carried out in this study increase the knowledge 39

about the relationship between peptide structure and iron-chelating activity and endorses the 40

promising role of whey protein hydrolyzates as functional ingredients in iron supplementation 41

treatments. 42

43

Keywords: whey protein; enzymatic hydrolysis; iron-binding peptide; Fe (III) immobilized 44

metal affinity chromatography; mass spectrometry 45

46

47

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SHORT COMMUNICATION 48

Iron constitutes one of the most essential trace elements in human nutrition. According 49

to the World Health Organization (WHO/FAO, 2004), iron deficiency represents one of the 50

most important nutritional disorders worldwide, which is especially common under states of 51

increased iron requirement, insufficient intake or decreased bioavailability. There has been 52

great interest over the years in the study of metal chelating peptides. Recent reports have 53

demonstrated improved iron-binding capacity or bioavailability for peptides from diverse 54

sources (Guo et al., 2014). 55

Whey, which contains around 20% of milk proteins, represents the major by-product 56

of cheese manufacturing process (Smithers, 2008). Although casein contains more sites and a 57

higher affinity to bind iron than whey proteins due to the presence of phosphoserine residues, 58

the binding of iron by whey proteins has also been described (Sugiarto et al., 2009). In a study 59

for the optimization of the iron-binding capacity of β-lactoglobulin (β-Lg) by enzymatic 60

hydrolysis, Zhou et al. (2012) reported that whole β-Lg exhibited the lowest capacity 61

compared with any of the hydrolyzates tested. Hydrolysis of whey proteins with different 62

enzymes has been reported to release peptides with iron-chelating capacity, e.g., hydrolyzates 63

prepared with Alcalase (Kim et al., 2007), Corolase®

PP (O'Loughlin et al., 2014) or papain 64

and Neutrase (Ou et al., 2010). However, only few iron-binding peptides have been 65

characterized and, to our knowledge, no whey-derived peptides have been yet identified. This 66

scarce knowledge about the relationship between peptide structure and iron-chelating activity 67

does not allow to anticipate empirical activity. 68

It is known the notable proteolytic activity of Alcalase, pancreatin, and Flavourzyme 69

on whey proteins compared with other enzymes (Kim et al., 2007). Recently, we found that 70

the fraction < 5 kDa from whey protein hydrolyzates obtained individually with these three 71

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enzymes, showed a marked iron-chelating capacity detected by an enhancement of iron 72

dialyzability (De la Hoz et al., 2014a). 73

The aim of the current study is the identification of the peptides with affinity for iron 74

in these hydrolyzates and the comparative analysis of total amino acid profile. The peptide 75

structure required to exhibit iron-binding properties is discussed. 76

Enzymatic hydrolyzates were prepared according to De la Hoz et al., (2014a), as result 77

of an experimental Rotatable Central Composite Design (2² RCCD) to optimize the degree of 78

hydrolysis. Iron-binding peptides were isolated from < 5 kDa fractions by using iron (III)-79

immobilized metal ion affinity chromatography [IMAC-Fe (III)] as described by De la Hoz et 80

al. (2014b). Peptides were identified by using reverse-phase high performance liquid 81

chromatography and tandem mass spectrometry (RP-HPLC-MS/MS) according to Martínez-82

Maqueda et al. (2013). Samples were injected at the protein concentration of 2 mg/mL for < 5 83

kDa fractions and 1 mg/mL for IMAC-Fe (III) eluted fractions and analyzed over two mass-84

to-charge (m/z) ranges: 100-1,500 and 100-3,000, selecting the molecular ion target of 750 85

and 1,500 (m/z) respectively. 86

For the determination of amino acid profile, sample cleanup was carried out by the use 87

of solid phase extraction in anion mixed mode. Samples suspended in 2% NH4OH (v/v) were 88

applied to Bond Elut PAX®

cartridges (Waters, Santa Clara, CA, US) and sequentially eluted 89

with methanol and 5% formic acid (v/v methanol). Samples with norleucine, as internal 90

standard, were subjected to acid hydrolysis with 6 M HCl at 110ºC for 21 h (controlled 91

atmosphere). A Biochrom 30 Amino Acid Analyzer (Amersham-Pharmacia Biotech) was 92

used to quantify amino acids after the separation by cation exchange chromatography and 93

postcolumn ninhydrin derivatization, according to Spackman et al. (1958). 94

Figure 1 shows the identified peptides from β-Lg, the major protein of bovine whey, in 95

IMAC-Fe (III)-bound fractions. Regardless of the enzyme employed, iron-binding peptides 96

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could be mainly grouped in two regions: β-Lg f(42-59) and β-Lg f(122-137), which enclosed 97

most of the identified peptides. This situation was especially significant in the case of 98

pancreatin, where no IMAC-Fe (III)-bound peptides from different regions were found. 99

Interestingly, these two domains are rich in negatively charged amino acids, such as, Glu and 100

Asp. In the region β-Lg f(42-59), 5 of the 18 amino acids are Glu or Asp while in the domain 101

β-Lg f(122-137) up to 6 of the 13 residues correspond to these negatively charged residues. 102

Figure 2 shows the α-lactalbumin (α-La)-derived peptides identified in the IMAC-Fe 103

(III)-bound fraction after hydrolysis with Alcalase, pancreatin, and Flavourzyme. It has to be 104

noted that both, Flavourzyme and pancreatin, render high negatively charged peptides from 105

the region α-La f(81-88) where five from the 8 residues corresponded to Asp. Enzymatic 106

treatments with Alcalase and pancreatin yielded a higher number of α-La IMAC-bound 107

peptides than that observed with Flavourzyme. 108

Additionally, the detailed characterization of < 5 kDa and IMAC-Fe (III)-derived 109

fractions is presented in the Supplementary Table 1 of the Appendix. It can be seen that a high 110

proportion of peptides identified in the fraction < 5 kDa of the hydrolysis with Flavourzyme 111

were bound to the IMAC-Fe (III) column. On the contrary, the ratio considerably decreased in 112

the hydrolyzates with the other two enzymes, especially with Alcalase. It is known that the 113

structure of β-Lg is very stable, in a compact globular conformation stabilized by two 114

intramolecular disulphide bonds, which restricts the action of certain enzymes under common 115

physicochemical conditions (Otte et al., 1997). This feature would explain the fact that there 116

are wide regions of the protein with scarce or null coverage. The broad endoproteinase 117

activity and substrate specificity of Alcalase and the endoproteinase and exopeptidase 118

activities of Flavourzyme could explain the presence of peptides from different β-Lg regions 119

(O'Keeffe & FitzGerald, 2014). Among the enzymatic treatments, the hydrolysis with 120

Flavourzyme showed the greatest variety of iron-binding peptides. 121

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The determination of amino acid profile was performed in order to provide 122

information about the most abundant amino acids in the IMAC-Fe (III)-bound fractions. 123

Figure 3 shows, for each enzyme treatment, the relative content of the different amino acids in 124

the parental (< 5 kDa) and IMAC-Fe (III)-derived fractions, which shows the enrichment of 125

certain amino acids by IMAC-Fe (III) chromatography. Despite the enzyme used to prepare 126

the hydrolyzate, the amino acids Asp and Glu were significantly enriched in the 127

chromatographic bound fractions, especially in those obtained with pancreatin and 128

Flavourzyme. A known handicap of the amino acid determination is the deamination of Asn 129

and Gln during sample preparation, being detected as Asp and Glu, respectively. However, it 130

is logical that the iron bound fractions are enriched in amino acids of acid character, based in 131

the results obtained by tandem mass spectrometry. These amino acids (Asp and Glu) achieved 132

the highest increases in IMAC-Fe (III)-bound fractions compared to the < 5 kDa hydrolyzate 133

with percentage differences up to 8.9 and 11.2 units for pancreatin, respectively. Moreover, 134

Pro showed an increase of 4.5 percentage units in the bound fraction respect to the 135

hydrolyzate obtained with Flavourzyme. In fact, Pro with a percentage of 10.9 constituted the 136

third most abundant amino acid in the IMAC-Fe (III)-bound fraction obtained from the 137

Flavourzyme hydrolyzate, which is also consistent with the results obtained by mass 138

spectrometry. In contrast, other amino acids exhibited a decrease in the iron-bound fraction. 139

Leu is the amino acid whose content was remarkably reduced in the bound fractions, with 140

decreases of 4.3, 7.6, and 7.5% respect to Alcalase, pancreatin, and Flavourzyme < 5 kDa 141

hydrolyzates, respectively. 142

These results agree with previous reports in the literature about the amino acid 143

composition of iron-binding peptides. The key role of Asp, Glu and Pro in the chelation of 144

iron by peptides is known. The enrichment of peptides in these amino acids has been related 145

with the enhancement of iron uptake by Caco-2 cell (Ou et al., 2010) and the positive effect of 146

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meat on nonheme iron bioavailability (meat factor) (Storcksdieck, et al., 2007). It is reported 147

that Asp and Glu form very stable iron chelates with a probable tridentate structure (Perrin, 148

1958) and Pro may contribute by the formation of stable complexes, supported by the special 149

cyclic nature of this amino acid (Nelson & Cox, 2004). Moreover, the stability of iron-Pro 150

complexes can be considered as an additional advantage since Pro-rich peptides show an 151

enhanced resistance to digestion enzymes (Yaron et al., 1993). 152

In conclusion, the identification of peptide sequences with iron-binding capacity from 153

whey proteins has been performed for the first time. Peptides were obtained by hydrolysis of 154

WPI with three different enzymes: Alcalase, pancreatin and Flavourzyme. Regardless the 155

enzyme used to prepare the hydrolyzates, the domains f(42-59) and f(125-137) from β-Lg 156

enclosed most of identified peptides in the chromatographic bound fraction. Iron-bound 157

peptide pools from IMAC-Fe (III) also exhibited common characteristics such as the 158

abundance of iron-chelating amino acids (Asp, Glu and Pro). Nevertheless, each enzyme 159

presented particular properties as specific proportion of certain amino acids and different 160

specificity on major whey proteins. The characterization of iron-binding peptides carried out 161

in this study increase the knowledge about the relationship between peptide structure and 162

iron-chelating activity and endorses the promising role of whey protein hydrolyzates as 163

functional ingredients in iron supplementation treatments. Additional studies of iron uptake 164

should be carried out in cellular and in vivo models to assess the capacity of these 165

hydrolyzates to improve iron bioavailability. 166

ACKNOWLEDGEMENTS 167

This work has received financial support from projects AGL2011-24643, FEDER-168

INNTERCONECTA-GALICIA (ENVELLEFUN), FP7-SME-2012-315349 (FOFIND), 169

Fundação de Amparo a Pesquisa de São Paulo (FAPESP) and IBEROFUN CYTED. E. C.-H. 170

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thanks to the University of Veracruz, Mexico, for the PROMEP/103.5/13/6408 scholarship 171

for support for PhD studies abroad. 172

REFERENCES 173

De la Hoz, L., V. S. Nunes da Silva, M. A. Morgano, and M. T. B Pacheco. 2014a. Small 174

peptides from enzymatic whey hydrolysates increase dialyzable iron. Int. Dairy J. 38: 145-175

147. 176

De la Hoz, L., A. N. Ponezi, R. F. Milani, V. S. Nunes da Silva, A. Sonia de Souza, and M. T 177

Bertoldo-Pacheco. 2014b. Iron-binding properties of sugar cane yeast peptides. Food Chem. 178

142: 166-169. 179

Guo, L., P. A. Harnedy, B. Li, H. Hou, Z. Zhang, X. Zhao, and R. J. FitzGerald. 2014. Food 180

protein-derived chelating peptides: Biofunctional ingredients for dietary mineral 181

bioavailability enhancement. Trends Food Sci. Tech. 37: 92-105. 182

Kim, S. B., I. S. Seo, M. A. Khan, K. S. Ki, M. S. Nam, and H. S. Kim. 2007. Separation of 183

iron-binding protein from whey through enzymatic hydrolysis. Int. Dairy J. 17: 625-631. 184

Martínez-Maqueda, D., B. Miralles, M. Ramos, and I. Recio. 2013. Effect of β-lactoglobulin 185

hydrolysate and β-lactorphin on intestinal mucin secretion and gene expression in human 186

goblet cells. Food Res. Int. 54: 1287-1291. 187

Nelson, D. L., and M. M. Cox. 2013. Chapter 5. Pages 157-189 in Lehninger Principles of 188

Biochemistry. W.H. Freeman Publishers, New York. 189

O'Keeffe, M. B., and R. J. FitzGerald. 2014. Antioxidant effects of enzymatic hydrolysates of 190

whey protein concentrate on cultured human endothelial cells. Int. Dairy J. 36: 128-135. 191

O'Loughlin, I. B., B. A. Murray, R. J. FitzGerald, A. Brodkorb, and P. M. Kelly. 2014. Pilot-192

scale production of hydrolysates with altered bio-functionalities based on thermally-denatured 193

whey protein isolate. Int. Dairy J. 34: 146-152. 194

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Otte, J., M. Zakora, K. B. Qvist, C. E. Olsen, and V. Barkholt. 1997. Hydrolysis of bovine β-195

lactoglobulin by various proteases and identification of selected peptides. Int. Dairy J. 7: 835-196

848. 197

Ou, K., Y. Liu, L. Zhang, X. Yang, Z. Huang, M. J. R. Nout, and J. Liang. 2010. Effect of 198

neutrase, alcalase, and papain hydrolysis of whey protein concentrates on iron uptake by caco-199

2 cells. J. Agr. Food Chem. 58: 4894-4900. 200

Perrin, D. D. 1958. The stability of complexes of ferric ion and amino-acids. J. Chem. Soc. 201

3125-3128. 202

Smithers, G. W. 2008. Whey and whey proteins-from 'gutter-to-gold'. Int. Dairy J. 18: 695-203

704. 204

Spackman, D. H., W. H. Stein, and S. Moore. 1958. Automatic recording apparatus for use in 205

the chromatography of amino acids. Anal. Chem. 30: 1190-1206. 206

Storcksdieck, S., G. Bonsmann, and R. F. Hurrell. 2007. Iron-binding properties, amino acid 207

composition, and structure of muscle tissue peptides from in vitro digestion of different meat 208

sources. J. Food Sci. 72: S19-S29. 209

Sugiarto, M., A. Ye, and H. Singh. 2009. Characterisation of binding of iron to sodium 210

caseinate and whey protein isolate. Food Chem. 114: 1007-1013. 211

WHO/FAO. 2004. Vitamin and mineral requirements in human nutrition. 2nd ed., Geneva, 212

Switzerland. 213

Yaron, A., F. Naider, and S Scharpe. 1993. Proline-dependent structural and biological 214

properties of peptides and proteins. Crit. Rev. Biochem. Mol. Biol. 28: 31-81. 215

Zhou, J., X. Wang, T. Ai, X. Cheng, H. Y. Guo, G. X. Teng, and X. Y. Mao. 2012. 216

Preparation and characterization of β-lactoglobulin hydrolysate-iron complexes. J. Dairy Sci. 217

95: 4230-4236. 218

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Figure legends 219

Figure 1. Schematic representation of peptides identified from bovine β-lactoglobulin in 220

fraction derived from iron (III)-immobilized metal ion affinity chromatography for different 221

enzymatic treatments (Alcalase, pancreatin, and Flavourzyme). Identification performed by 222

reverse-phase high performance liquid chromatography and tandem mass spectrometry. 223

Figure 2. Schematic representation of peptides identified from bovine α-lactalbumin in 224

fraction derived from iron (III)-immobilized metal ion affinity chromatography for different 225

enzymatic treatments (Alcalase, pancreatin, and Flavourzyme). Identification performed by 226

reverse-phase high performance liquid chromatography and tandem mass spectrometry. 227

Figure 3. Amino acid content determined by Biochrom 30 Amino Acid Analyzer. Data 228

expressed, for each amino acid, as mean and standard deviation of the percentage (%) of the 229

sum of all amino acid identified (n=2). Asn and Gln detected as Asp and Glu, respectively, 230

due to their deamination during the preparation step. Trp completely degraded during the 231

analysis. WPH specifies the fraction < 5 kDa and IMAC-Fe (III), the iron (III)-immobilized 232

metal ion affinity chromatography fraction. 233

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... ... ...

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Asp/Asn Thr Ser Glu/Gln Gly Ala Cys Val Met Ile Leu Tyr Phe His Lys Arg Pro

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Supplementary Table 1. Identified peptides from bovine β-lactoglobulin and α-lactalbumin 1

for different enzymatic treatments (Alcalase, pancreatin, and Flavourzyme). Identification 2

performed by reverse-phase high performance liquid chromatography and tandem mass 3

spectrometry. WPH specifies the fraction <5 kDa and IMAC, the iron (III)-immobilized metal 4

ion affinity chromatography [IMAC-Fe (III)] fraction. 5

β-LACTOGLOBULIN

FRAGMENT AMINOACID SEQUENCE

THEORETICAL

MASS

ALCALASE PANCRETIN FLAVOURZYME

WPH IMAC WPH IMAC WPH IMAC

01 - 05 LIVTQ 572,35 • 02 - 05 IVTQ 459,27 • 06 - 10 TMKGL 548,30 • 08 - 13 KGLDIQ 672,38 • 08 - 20 KGLDIQKVAGTWY 1477,79 • • 26 - 32 ASDISLL 717,39 • 27 - 32 SDISLL 646,35 • 42 - 58 YVEELKPTPEGDLEILL 1957,03 • • 42 - 57 YVEELKPTPEGDLEIL 1843,95 • 42 - 56 YVEELKPTPEGDLEI 1730,86 • 42 - 55 YVEELKPTPEGDLE 1617,78 • • 42 - 47 YVEELK 777,40 • 43 - 58 VEELKPTPEGDLEILL 1793,97 • •

43 - 57 VEELKPTPEGDLEIL 1680,88 • • • •

43 - 56 VEELKPTPEGDLEI 1567,80 • • • •

43 - 55 VEELKPTPEGDLE 1454,71 • • • • • •

43 - 54 VEELKPTPEGDL 1325,67 • • • •

43 - 53 VEELKPTPEGD 1212,59 • • 43 - 51 VEELKPTPE 1040,54 • 43 - 49 VEELKPT 814,44 • 44 - 58 EELKPTPEGDLEILL 1694,90 • • 45 - 55 ELKPTPEGDLE 1226,60 • • 46 - 57 LKPTPEGDLEIL 1323,73 • 46 - 55 LKPTPEGDLE 1097,56 • • • 46 - 51 LKPTPE 683,39 • 47 - 58 KPTPEGDLEILL 1323,73 • •

47 - 57 KPTPEGDLEIL 1210,64 • •

47 - 56 KPTPEGDLEI 1097,56 • • • •

47 - 55 KPTPEGDLE 984,48 • • • •

47 - 54 KPTPEGDL 855,43 • •

49 - 59 TPEGDLEILLQ 1226,64 • •

49 - 58 TPEGDLEILL 1098,58 •

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49 - 55 TPEGDLE 759,33 • • 50 - 57 PEGDLEIL 884,45 • 50 - 56 PEGDLEI 771,36 • 52 - 56 GDLEI 545,27 • 71 - 74 IIAE 444,26 • 73 - 86 AEKTKIPAVFKIDA 1529,88 • 73 - 85 AEKTKIPAVFKID 1458,84 • •

73 - 84 AEKTKIPAVFKI 1343,82 • •

73 - 82 AEKTKIPAVF 1102,64 • •

73 - 81 AEKTKIPAV 955,57 • •

73 - 80 AEKTKIPA 856,50 • •

73 - 79 AEKTKIP 785,46 • •

74 - 82 EKTKIPAVF 1031,60 • 74 - 80 EKTKIPA 785,46 • 75 - 82 KTKIPAVF 902,56 • • 76 - 82 TKIPAVF 774,46 • 76 - 80 TKIPA 528,33 • 77 - 82 KIPAVF 673,42 • 83 - 87 KIDAL 558,34 • 88 - 93 NENKVL 715,39 •

96 - 102 DTDYKKY 931,43 • • • •

96 - 100 DTDYK 640,27 • 97 - 102 TDYKKY 816,40 • •

97 - 101 TDYKK 653,34 • •

110 - 116 SAEPEQS 746,31 • 111 - 116 AEPEQS 659,27 • 122 - 131 LVRTPEVDDE 1171,57 • • 122 - 127 LVRTPE 713,41 • • 123 - 134 VRTPEVDDEALE 1371,65 • 123 - 131 VRTPEVDDE 1058,49 • 123 - 127 VRTPE 600,32 • 124 - 135 RTPEVDDEALEK 1400,68 • •

124 - 129 RTPEVD 715,35 • • 125 - 137 TPEVDDEALEKFD 1506,67 • 125 - 136 TPEVDDEALEKF 1391,65 • •

125 - 135 TPEVDDEALEK 1244,58 • • • • 125 - 134 TPEVDDEALE 1116,48 • • 125 - 132 TPEVDDEA 874,36 • • • • 125 - 131 TPEVDDE 803,32 • • 128 - 134 VDDEALE 789,34 • • 135 - 139 KFDKA 607,33 • • 137 - 144 DKALKALP 854,52 • 137 - 141 DKALK 573,35 •

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137 - 140 DKAL 445,25 • 142 - 145 ALPM 430,22 • 146 - 150 HIRLS 624,37 • 146 - 149 HIRL 537,34 • • 149 - 154 LSFNPT 677,34 • • 152 - 158 NPTQLEE 829,38 • •

155 - 159 QLEEQ 645,29 • • 6

7

α-LACTALBUMIN

FRAGMENT AMINOACID SEQUENCE

THEORETICAL

MASS

ALCALASE PANCREATIN FLAVOURZYME

WPH IMAC WPH IMAC WPH IMAC

01 - 05 EQLTK 617.34 •

07 - 12 EVFREL 791.42 •

10 - 15 RELKDL 772.44 • •

13 - 18 KDLKGY 722.40 • •

14 - 26 DLKGYGGVSLPEW 1419.70

• •

19 - 27 GGVSLPEWV 942.48

•

19 - 26 GGVSLPEW 843.41

•

46 - 52 DSTEYGL 783.33

•

50 - 53 YGLF 498.25 •

63 - 70 DDQNPHSS 898.34 •

79 - 90 KFLDDDLTDDIM 1439.65 •

80 - 90 FLDDDLTDDIM 1311.55

•

80 - 89 FLDDDLTDDI 1180.51

•

81 - 90 LDDDLTDDIM 1164.49

• •

81 - 89 LDDDLTDDI 1033.45

• •

81 - 88 LDDDLTDD 920.36

• •

82 - 90 DDDLTDDIM 1054.40

• •

82 - 89 DDDLTDDI 920.36

• •

82 - 88 DDDLTDD 807.28

• •

94 - 103 KILDKVGINY 1161.68 • •

94 - 102 KILDKVGIN 998.61 •

94 - 99 KILDKV 714.46 • •

97 - 103 DKVGINY 807.41 •

97 - 102 DKVGIN 644.35 •

•

8

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Documents

For Peer Review - Digital CSICdigital.csic.es/bitstream/10261/150457/4/iron-binding... · 2018-09-19 · For Peer Review 1 1 INTERPRETIVE SUMMARY 2 Short communication: Identification