Development of non-invasive stress biomarkers in octopuses

Rachel Thompson
June 1, 2009

Abstract

Octopuses are an important part of the Pacific Northwest ecosystem. Recently,

local populations have been experiencing declines due to pollution and large-scale

climate processes. Non-invasive sampling techniques were developed in an attempt to

characterize the physiological condition of adult octopuses in a controlled environment.

Potential biomarkers of stress (proteins in epidermal mucus and behavior patterns) were

identified, and could be useful in identifying a stressed state in an octopus in the wild or

in captivity. Larval developmental patterns were established by analysis of the expression

of two important developmental genes, orthodenticle-like protein and hedgehog. This

study serves to provide researchers and aquarists with baseline physiological data that

could be used to identify a stressed state in an adult octopus, or an altered developmental

pattern in larvae, using techniques that are less invasive than hemolymph or tissue

sampling.

Introduction

Octopuses are advanced invertebrates with a strong tie with the Pacific Northwest.

Two local species are the Red Octopus (Octopus rubescens) and the Giant Pacific

Octopus (Enteroctopus dofleini). Both are benthic species, with ranges along the Eastern

Pacific Ocean from Mexico to Alaska. Recently, populations have been declining due to

several factors, including pollution and large-scale climate processes affecting the Puget

Sound region (Rigby et al. 2005, Villanueva and Norman 2008). Octopus species are

important prey items for higher predators in Puget Sound. Their conservation is essential

to the maintenance of the region’s trophic structure (Onthank and Cowles 2008).
 Octopuses are oviparous organisms that brood and care for their eggs during

development (Kaneko et al. 2006). Red Octopus larvae hatch approximately 6 to 8 weeks

after they are laid, and enter the water column as planktonic predators (von Boletzky

2003). The timing and success of development is dramatically affected by environmental

variables, such as changes in temperature and exposure the chemicals (Clarke et al.

2009). In a laboratory setting, increased temperatures (to 23 degrees Celsius from 18

degrees Celsius) accelerated the development of octopus hatchlings, and shortened their

life spans by up to 20% (Forsythe et al. 1988). The effect on adult octopuses has yet to be

determined. However, as cephalopods are poikilothermic organisms, their physiology

during all life stages is dramatically influenced by water temperature. The challenge lies

in identifying and quantifying these influences.

Responses to stress in octopuses can be obvious, such as color changing when

faced by a predator, but indicators of chemical or temperature stress are not often visual.

Stress can impair immune function in many invertebrates, leading to decreased disease

resistance (Malham et al. 2003). The cephalopod immune system consists of humoral and

cellular mechanisms (Ford 1992). The common stress response of animals is intended to

maintain homeostasis when threatened by an environmental change. In cephalopods, this

response releases catecholamines into the hemolymph, which shut down body functions

related to growth, reproduction and immunity with the intent of allocating energy

resources towards more immediate concerns (Malham et al. 2002). Specific effects of

stressors, such as exposure to air, include decreased hemocyte counts, and a decrease in

phagocytotic activity in those remaining cells. Since immune defenses are down-

regulated in this manner while the animal is in a stressed condition, it is left more

susceptible to disease.
 The introduction of temperature and chemical stressors can have significant

effects on the physiology of aquatic organisms, including but not limited to, disruption of

development, reproduction and growth (Zala and Penn 2004). It has also been found that

exposure to chemical pollution, specifically endocrine-disrupting chemicals (EDCs), such

as those found in urbanized areas of the Puget Sound, can result in abnormal behavior in

vertebrates (Zala and Penn 2004). EDCs can have adverse effects on a range of behaviors

that are controlled by hormones, including reproductive and sexual behavior, activity

level, aggression, communication and motivation (Zala and Penn 2004). It is therefore

likely that analyzing changes in behavior patterns could be used as an indicator, or

biomarker, for detecting harmful environmental contaminants (Zala and Penn 2004). It

has been suggested that behavior might be a more useful indicator or “biomarker” than

standard assays. Behavioral assays are non-invasive, inexpensive and potentially more

powerful than other methods, since behavior is the outcome of many complex

developmental and physiological processes (Zala and Penn 2004). On the other hand,

behavior can be difficult to measure and highly variable. The combination of behavioral

and physiological data could provide a comprehensive method for characterizing the

octopus stress response.

Gene expression patterns are reflective of the timing of particular events in

development. Many developmental genes are highly conserved in the animal kingdom

(Ingham and McMahon 2001). Two important genes are those that code for orthodenticle-

like protein and hedgehog, both of which play critical roles in body patterning and

morphogenesis. Analysis of gene expression changes can provide insight into the

physiological state of an organism. For example, gene expression “rhythms” in rocky

intertidal mollusks allow them to adapt to low-tide heat stress events (Gracey et al. 2008).
Since developing larvae are particularly vulnerable to temperature stress, changes in gene

expression could be dramatic and interesting.

The overall goal of this project is to develop techniques to characterize the stress

response of octopus species (O. rubescens and E. dofleini) using epidermal mucus and

developmental gene expression patterns. Information is lacking on stress response in

octopuses and more importantly, non-lethal, non-invasive means to detect it.

Understanding the physiological response of octopuses to stress could be valuable for

conservation purposes, predicting effects of environmental stress on local food chains,

and simply gaining a better understanding of the lives of these popular, local species. It

would be highly useful to isolate biological markers from epidermal surfaces rather than

internal body tissues. A method such as this would allow the animal to live, as well as

save time and cost of sampling. Potential behavioral and molecular stress biomarkers will

be identified in adults using these non-invasive methods, and the gene expression patterns

of two important developmental genes will be analyzed in larvae. Ultimately, this study

will serve to establish a baseline physiological data set for both adult and larval Red

Octopuses

Methods

Epidermal mucus collection

Epidermal mucus was collected from a female Red Octopus (O. rubescens). Three

approaches were attempted to determine the most effective sampling procedure. The first

approach was to collect mucus using a plastic transfer pipet with the octopus still

submerged in the water. The second was to remove the octopus from the water and place

it into a sampling container. The third approach was to remove the liquid left in the

bottom of the sampling container after the octopus was removed. This liquid theoretically
contained a mixture of seawater and mucus.

Protein analysis

Samples were combined with SDS, heated to 100 degrees Celsius for 10 minutes, and run

on an SDS PAGE gel (Pierce 4-20% Tris-Hepes) for 45 minutes at 150 Volts. Gels were

stained with Coomassie Blue Stain for approximately one hour. It was determined that

silver staining was more sensitive to the detection of protein bands, and this procedure

was used for later staining procedures. Non-invasive techniques were used to identify

possible protein biomarkers of stress. Proteins were identified using two approaches.

Discovery-based approach: Mass spectroscopy

Prominent bands were excised from a gel containing samples collected using each of the

three techniques attempted. Bands were chosen based on intensity, pattern and whether or

not they were present in all samples. The excised bands were de-stained according to

Invitrogen protocol. Trypsin digestion was performed according to the Goodlett lab

protocol. The digested proteins were sequenced using mass spectroscopy, and a list of

proteins and short sequences found in the bands was received. Stress or immune-related

proteins were noted, and investigated to determine possible use as biomarkers of stress.
Lanes: 1 2 3 4 5 6 7 8

Figure 1. Protein gel of epidermal mucus samples collected using various sampling

techniques. Lanes 2,3, 7 and 8 contain a 1:1 ratio of mucus collected from the sampling

container and 2X SDS. Lane 4 contains mucus collected underwater. Lanes 5 and 6

contain mucus collected with octopus in the sampling container.

Targeted approach: Western blotting

Heat shock protein 70 (HSP 70) was targeted using the Western blot technique. It was

determined that mucus samples contained protein concentrations too low for the HSP 70

antibodies to detect. Leg tissue samples were collected weekly from this point on. Protein

was extracted from the leg tissue using 500 uL of Cell Lytic. A Bradford protein assay

was performed in order to determine the protein concentration of the extracted samples. A

total of seven leg tissue protein samples containing 9.7 ug of protein were run on an SDS

PAGE gel for 45 minutes at 150 Volts. A Western blot was done using an Invitrogen

Western Breeze (anti-mouse) kit and the anti-HSP 70 antibody. The Western was then

developed according to the Western Breeze protocol.

Behavioral assessment

A web-cam was installed outside of the octopus aquarium. This provided a 24

hour video feed of its activity. Time lapse videos were available online. The octopus’

behavior at randomly determined time intervals was categorized by certain activities

characteristic of varying levels of behavior. These included swimming, crawling, and

remaining stationary on the side of the aquarium. Prior to imposing environmental stress,

baseline behavior was categorized. An online viewer survey was associated with the live

video feed. Viewers were able to check boxes corresponding to these same activity

categories, the results of which were imported into an Excel spreadsheet for later

analysis.

Larval gene expression

Egg samples were collected weekly. RNA was extracted using the Tri-Reagent

isolation protocol. A Bradford nucleic acid assay was performed in order to determine the

RNA concentration of the samples. cDNA was made by reverse transcription. Primers for

two developmental genes were designed, orthodenticle-like protein (OTX, Octopus

bimaculoides) and hedgehog (HH, Octopus bimaculoides). Real-Time PCR was done

using an Opticon system. Data was analyzed according to Miner.

Results

Success varied in relation to the sampling technique used. The underwater

sampling method resulted in mucus samples that were too dilute with seawater to produce

bands after gel electrophoresis (Figure 2). Samples collected from the sampling container

had sufficient protein concentrations to produce easily visible bands. Mucus pipetted
directly from the epidermal surface produced bands similar in intensity and pattern to the

samples collected from the sampling container.

Lanes: 1 2 3 4 5 6 7

Figure 2. Mucus samples collected using three sampling techniques were visualized using

gel electrophoresis. Lanes 2,3, 6 and 7 contain mucus collected from the sampling

container. Lane 4 contains mucus collected underwater. Lane 5 contains mucus collected

with the octopus removed from the water.

Identification of protein biomarkers

 Discovery-based approach

Proteins present in the excised bands were identified using mass spectroscopy.

Table 1. Representative mass spectroscopy results after trypsin digestion. See appendix for

complete list of mass spectroscopy results.

Protein Total Description Peptide sequence

number
of
peptides
CAS1_BOVIN 20 (P02662) YLGYLEQ
Alpha-S1-
casein
precursor

LACB_BOVIN 3 (P02754) Beta- VLVLDTDYK

lactoglobulin
precursor

ACT_HYDAT,ACTC_STYPL 2 (Q00215) GYSFTTTAER

Actin,
cytoplasmic

CASB_BOVIN 25 (P02666) Beta- DM[147]PIQAF

casein
precursor
STAT_HUMAN 14 (P02808) FGYGYGPYQPVPEQPLYPQPYQPQY
Statherin QQYTF
precursor
VIME_HUMAN,VIME_PANTR 111 (P08670) ILLAELEQLK
Vimentin
PALA_EMENI 2 (P79020) pH- DDISSALVR
response
regulator
protein
DNAK_BLOFL 1 (Q7VQL4) HSQVFSTAEDNQSAVTIHVLQGER
Chaperone
protein dnaK
(Heat shock
protein 70)

These lists of proteins were sorted through and condensed into a short list of

proteins possibly related to stress and immune function. They were thus assumed to be
potentially useful as biomarkers of stress (Lahov and Regelson 1996, Barak et al. 2005).

Table 2. Selected proteins identified from epidermal mucus samples by mass spectroscopy

with functions related to stress or immune function. These proteins have potential use as

biomarkers.

Protein Function
Casein Antioxidant peptide, radical scavenging
activity, inhibits growth of E. coli
Heat Shock Protein 70 (Chaperone protein dnaK) Chaperone and monitor for other proteins,
maintains conformations, disposal of
degraded proteins
Dermcidin Antimicrobial, limits skin infection by
potential pathogens

Targeted protein analysis

The antibody for Heat Shock Protein identified the presence of the protein in the
extracted leg tissue samples (Figure 3).

Lanes: 1 2 3 4 5 6 7 8 9 10 11 12

Figure 3. Nitrocellulose membrane following Western blotting. The dark marker indicates

the presence of HSP 70 in the leg tissue samples. Lanes 2 through 8 contain 9.7 ug of leg

tissue protein, loaded into wells in chronological order. The lane 2 sample was collected

on 10/24/2008, the lane 8 sample was collected on 4/1/2009. Lanes 9 through 12 contain

20 ul of mucus collected from the skin with the octopus removed from the water. The

lane 9 sample was collected on 1/28/2009 and the lane 12 sample was collected on

4/8/2009.

Heat shock protein is known to be produced in increased amounts during stressful

conditions. Therefore, HSP 70 could be a useful biomarker for identifying stress in

octopus species.

Identification of behavior biomarkers

 Crawling
Swimming
On Side of Aquarium

Figure 4. Baseline activity patterns were established after time lapse video analysis.

Prior to experimentation, behavior patterns were categorized. The octopus was

quite active. At the three randomly determined time intervals, it was often crawling and

swimming in the aquarium. 40% of time was spent crawling on the substrate or side of

aquarium, 40% of time was spent swimming, and a minimal amount of time (20%) was

spent stationary on the side of the aquarium (Figure 3). These patterns will serve as a

baseline level of activity (control) to be used for comparison after stressful conditions are

imposed.
 C rawling
Brooding
On Side of Aquarium
No Data

Figure 5. The activities of the octopus were categorized after eggs were laid.

After the eggs were laid, behavior was again categorized to determine the impact

of post-reproductive senescence (a biological rather than environmental stressor) on

behavior. The general behavioral pattern showed a shift from active crawling and

swimming to stationary activities. Immediately after the eggs were laid, the female

octopus began a constant routine of brooding. Brooding entailed positioning itself on top

of the eggs and blowing water through the siphon to oxygenate the egg strands.

Approximately 90% of time was spent brooding the eggs on the side of the aquarium,

while 2% of time was spent crawling. For 8% of the time, data was not available due to

camera malfunction (Figure 4).

Larval gene expression

Expression (Fold over Minimum)

1000000

100000

10000

1000

100

10

1
9 09 09 09 09 09 09 09 09 09 09
/0 4/ / / / 4/ / / / 1/ 8/
28 2/ 11 18 25 3/ 11 18 25 4/ 4/
1/ 2/ 2/ 2/ 3/ 3/ 3/

Date

Figure 6. Orthodenticle-like protein expression (log of fold over minimum) in octopus

eggs collected weekly from 1/28/09 to 4/12/09.

There was a 100,000-fold increase in expression of the gene for orthodenticle-like

protein (OTX) over the course of the 10 week sampling period. There was zero gene

expression on 1/28/09, which was the day the eggs were laid. A minimum expression

(Plotted as zero in Figure 5) was quantified on 2/4/09. From 2/4/09 to 4/12/09, the

expression of OTX increased fairly steadily up to its maximum value of 100,000 fold

over minimum.
 1000

Expression (Fold over Minimum)

100

10

/0
9 09 0 9 0 9 0 9 09 /0
9
/0
9
/0
9 09 09
28 4/ 1/ 8/ 5/ 4/ 11 18 25 1/ 8/
1/ 2/ 2/
1
2/
1
2/
2 3/ 3 / 3 / 3 / 4/ 4/

Date

Figure 7. Hedgehog expression (log of fold over minimum) in octopus eggs collected

weekly from 1/28/09 to 4/12/09.

The hedgehog gene was expressed relatively late in development. A minimum

expression was quantified on 3/25/09, with zero expression up until that date. Maximum

expression was reached on 4/8/09, which represents an approximate 300-fold increase

over minimum over the course of several weeks. Expression declined after 4/8/09, and

reached a final value of less than a 100-fold increase over minimum at the end of the

sampling period.
Discussion

The development of non-invasive sampling techniques was fairly successful. 3

methods were tested, and results varied. It was determined that collecting epidermal

mucus from the sampling container, and directly from the epidermal surface with the

octopus removed from the water were the two most useful methods. In this sense,

usefulness was measured by the intensity and number of protein bands present after

protein gel electrophoresis. Mucus samples collected underwater were not useful, as no

bands were visible. Therefore, it is likely that collecting mucus from the skin and from

the sampling container could be useful techniques as a first step to identifying protein

biomarkers. Both of these methods were minimally invasive. Stress was imposed on the

octopus during both procedures, as the animal was removed from the water and was

exposed to air for approximately 5 minutes. This air exposure could also introduce

thermal stress, as the temperature of the air was approximately 10 degrees higher than the

temperature of the aquarium water. Therefore, it is possible that stress-related proteins

could be present in the control samples. Differences in the expression of many proteins

will have to be measured, rather than simply noting the presence of one or two stress-

related proteins in the experimental samples.

The non-invasive methods developed were used prior to identification of proteins.

The discovery-based approach provided an extensive list of proteins after mass

spectroscopy sequencing. Several interesting stress and immune related proteins were

present, and further investigation into their functions identified them as possible

biomarkers. Alpha, beta, and kappa caseins composed a large percentage of the proteins

identified. Caseins are milk proteins that function as antioxidants. Short casein peptides

participate in various biological activities. Some function as immunomodulators

(immunostimulators in cows and humans) (Fiat and Jolles 1988). Others have radical

scavenging activity (Clausen et al. 2009) and have been found to inhibit the growth of

E.coli, staphylococcus, and streptococcus bacteria (Lahov and Regelson 1996). It is

possible that caseins with similar functions could be found in octopus species. Exposure

to bacterial pathogens would certainly result in casein production. It is possible that

thermal or chemical stress could induce the production of casein proteins as part of a

stress response.

Another interesting protein with similar function is dermcidin. Dermcidins are

potent antimicrobial proteins that defend against pathogenic microorganisms including S.

aureus and E. coli (Barak et al. 2005). It is likely that dermcidin production would

increase when faced with a pathogenic stressor, and possible that thermal or chemical

stress could induce a response.

Heat shock protein 70 was detected in epidermal mucus samples. Heat shock

proteins are the most conserved proteins in both prokaryotes and eukaryotes (Schmitt et

al. 2007). They accumulate in cells exposed to thermal or other stressful stimuli. They

function as molecular chaperones and help cells to survive potentially lethal conditions,

as well as regulate apoptosis (Didelot et al. 2006). Heat shock proteins assist other

proteins in maintaining correct conformations during environmental stress, and transport

degraded proteins to proteasomes for disposal (Schmitt et al. 2007). Because heat shock

protein 70 is highly conserved and directly related to stress response, it was suitable for a

targeted-protein analysis using the Western blot technique. HSP 70 was detected by its

antibody in protein extracted from octopus leg tissue under the control sampling

conditions. It is potentially the most useful protein biomarker identified, as its function is

well-known and applicable to an experiment in which thermal stress is imposed.

However, the sampling technique required to perform a Western blot was more invasive

than the collection of epidermal mucus for mass spectroscopy. A Western blot was

attempted using epidermal mucus, but it was determined that the protein concentrations

of the samples were too low to get a clear result. It is also possible that HSP 70 is not

found in mucus. Small leg tissue samples were collected, which seemed to have little

effect on the well-being of the octopus. Its activity level remained high after the

procedure. Octopus leg tissue regenerates after damage, and after several days the leg

began to re-grow. As this procedure did not require anesthesia and did not cause long-

term damage to the octopus, it is still considered to be minimally invasive.

The techniques developed to analyze and categorize octopus behavior provided a

general, baseline pattern of daily activity. The difference between the activity patterns

before and after egg-laying was dramatic. The most obvious change was a shift from

active swimming and crawling to near constant brooding. The shift is a clear indicator of

the level of parental care provided by female octopuses to their young. The technique of

categorizing behavior based on time-lapse videos at randomly determined intervals could

be useful in evaluating the effect of environmental stress on octopus physiology.

Behavior is a direct result of physiological condition (Zala and Penn 2004). Behavioral

analysis is practical for many reasons. It is inexpensive, comprehensive and most

importantly for this study, non-invasive.

Future work would incorporate a controlled experiment to test whether the

production of casein, dermcidin, and heat shock protein 70 differs between control (non-

stressed) and experimental (stressed) conditions. The baseline physiological data could be

used for comparison purposes. This experiment would also validate the effectiveness of

the non-invasive sampling techniques developed.

The analysis of gene expression in octopus paralarval development revealed interesting

patterns. The expression patterns of the two genes investigated, orthodenticle-like protein

(OTX) and hedgehog (hh), varied greatly over time.

Orthodenticle-like protein is a DNA binding protein with the essential function of

regulating head and central nervous system development (Klein and Li 2002). OTX

proteins are widespread throughout the animal kingdom, and have evolved specialized

roles in some taxonomic groups. OTX appears to be expressed early in O. rubescens

development. This could indicate that morphogenesis of the anterior neural structures of

octopuses occurs close to the onset of development.

The hedgehog gene family has been found in vertebrates as well as several

invertebrate taxa, including mollusks. Hedgehog is expressed in both embryonic and

adult tissues, where it mediates development, growth, patterning and morphogenesis

(Grimaldi et al. 2007). Several studies have shown that hedgehog genes are highly

conserved in invertebrates (Ingham and McMahon 2001). Grimaldi et al. (2007) showed

that the hedgehog pathway is involved in the differentiation of striated muscle fibers in

the cuttlefish (Sepia officinalis) mantle. It is possible that hedgehog could also be

involved in muscle differentiation of octopus, since cuttlefish and octopuses are in the

same phylum. Hedgehog is expressed later in development (relative to OTX), which

could indicate that it is involved with patterning and morphogenesis at later stages of

development. This could be the time frame in which muscle fiber development occurs.

However, it is possible that hedgehog was expressed at low levels throughout

development, but the concentration was too low to be detected by the Real-Time PCR

equipment.
 Further investigation is necessary to determine the exact roles of orthodenticle-

like protein and hedgehog in octopus larval development. General expression patterns

were identified, including timing during development and relative increase in expression

over time. These increases in expression are likely due to a specific developmental event,

such as brain morphogenesis in the case of orthodenticle-like protein, or muscle fiber

differentiation in the case of hedgehog. These data could be used for comparison

purposes, possibly to identify changes in gene expression due to particular stressors, such

as temperature. Future work would involve mapping gene expression in specific body

regions and at specific times to determine the correlation between expression and the

development of organs.

The overall goal of this project was to determine if the stress-response of adult

octopuses can be characterized using non-invasive methods. This limited the scope of

possible techniques. A genetic analysis would have required tissue or hemolymph

samples which are more difficult to obtain, and involve procedures that are often harmful

to the animal. For this reason, institutions such as aquariums may value non-invasive

methods over hemolymph or tissue collection. Collecting epidermal mucus was fast,

inexpensive, and relatively harmless. The techniques used provided valuable information

about the protein composition of mucus, and sequencing identified several proteins that

may be related to stress-response. Identifying a stressed physiological state in a particular

animal could allow aquariums to ensure that the conditions of their exhibits do not

negatively affect the animals. Since senescence is a time period characterized by high

levels of stress and physiological change, these non-invasive methods could allow

researchers and aquarists to predict the onset of senescence, and therefore reproduction
and subsequent death.

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Appendix Group Protein Protein Percent Numbe Total Percent Description Peptide Sequence Precurs
Probabili Probabili Covera r of Numbe Share or Ion
ty ty ge Unique r of of Charge
Peptid Peptid Spectru
es es m ID’s
1 CAS1_BOV 1 26.2 12 20 3.53 (P02662) YLGYLEQ 1
IN Alpha-S1-
casein
precursor
1 CAS1_BOV 1 26.2 12 20 3.53 (P02662) EPMIGVNQELAYFY 2
IN Alpha-S1- PELFR
casein
precursor
1 CAS1_BOV 1 26.2 12 20 3.53 (P02662) EPM[147]IGVNQE 2
IN Alpha-S1- LAYFYPELFR
casein
precursor
1 CAS1_BOV 1 26.2 12 20 3.53 (P02662) FFVAPFPEVFGK 2
IN Alpha-S1-
casein
precursor
1 CAS1_BOV 1 26.2 12 20 3.53 (P02662) HQGLPQEVLNENL 2
IN Alpha-S1- LR
casein
precursor
1 CAS1_BOV 1 26.2 12 20 3.53 (P02662) IGVNQELAYFYPEL 2
IN Alpha-S1- FR
casein
precursor
1 CAS1_BOV 1 26.2 12 20 3.53 (P02662) YLGYLEQLLR 2
IN Alpha-S1-
casein
precursor
1 CAS1_BOV 1 26.2 12 20 3.53 (P02662) EPMIGVNQELAYFY 3
IN Alpha-S1- PELFR
casein
precursor
1 CAS1_BOV 1 26.2 12 20 3.53 (P02662) EPM[147]IGVNQE 3
IN Alpha-S1- LAYFYPELFR
casein
precursor
1 CAS1_BOV 1 26.2 12 20 3.53 (P02662) HQGLPQEVLNENL 3
IN Alpha-S1- LR
casein
precursor
Group Protein Protein Percent Numbe Total Percent Description Peptide Sequence Precurs
Probabili Probabili Covera r of Numbe Share or Ion
Group Protein Protein Percent Numbe Total Percent Description Peptide Sequence Precurs
Probabili Probabili Covera r of Numbe Share or Ion
ty ty ge Unique r of of Charge
Peptid Peptid Spectru
es es m ID’s
1 CAS2_BOV 1 23.4 5 6 1.06 (P02663) PITPTLNR 1
IN Alpha-S2-
casein
precursor
1 CASB_BO 1 29 12 19 3.29 (P02666) YPVEPFTESQ 1
VIN Beta-casein
precursor

1 CASB_BO 1 29 12 19 3.29 (P02666) DMPIQAFLLYQEPV 2

VIN Beta-casein LGPVR
precursor
1 CASB_BO 1 29 12 19 3.29 (P02666) PIQAFLLYQEPVLGP 2
VIN Beta-casein VR
precursor
DM[147]

1 CASB_BO 1 29 12 19 3.29 (P02666) FQSEEQQQTEDEL 2

VIN Beta-casein QDK
precursor
1 CASB_BO 1 29 12 19 3.29 (P02666) LLYQEPVLGPVR 2
VIN Beta-casein
precursor
1 CASB_BO 1 29 12 19 3.29 (P02666) DMPIQAFLLYQEPV 3
VIN Beta-casein LGPVR
precursor
1 CASB_BO 1 29 12 19 3.29 (P02666) PIQAFLLYQEPVLGP 3
VIN Beta-casein VR
precursor
DM[147]
1 CASB_BO 1 29 12 19 3.29 (P02666) IHPFAQTQSLVYPFP 2
VIN Beta-casein GPIPN
precursor

Group Protein Protein Percent Numbe Total Percent Description Peptide Sequence Precurs
Probabili Probabili Covera r of Numbe Share or Ion
ty ty ge Unique r of of Charge
Peptid Peptid Spectru
es es m ID’s
1 CASB_BO 1 29 12 19 3.29 (P02666) PIQAFLLYQEPVLGP 2
VIN Beta-casein VR
Group Protein Protein Percent Numbe Total Percent Description Peptide Sequence Precurs
Probabili Probabili Covera r of Numbe Share or Ion
ty ty ge Unique r of of Charge
Peptid Peptid Spectru
es es m ID’s
1 LACB_BOV 1 16.3 3 4 0.68 (P02754) VLDTDYK 1
IN,LACB_B Beta-
UBBU lactoglobulin
precursor
(Beta-LG)
(Allergen Bos
d 5),(P02755)
Beta-
lactoglobulin
precursor
(Beta-LG)
1 STAT_HUM 1 54.8 5 14 2.42 (P02808) FGYGYGPYQPVPE 3
AN Statherin QPLYPQPYQPQYQ
precursor QYTF
1 STAT_HUM 1 54.8 5 14 2.42 (P02808) IGRFGYGYGPYQPV 3
AN Statherin PEQPLYPQPYQPQY
precursor QQYTF

1 STAT_HUM 1 54.8 5 14 2.42 (P02808) RIGRFGYGYGPYQP 3

AN Statherin VPEQPLYPQPYQPQ
precursor YQQYTF
1 TRY1_RAT 1 8.1 2 3 0.51 (P00762) LGEHNINVLEGDE 2
Anionic QFINAAK
trypsin-1
precursor (EC
3.4.21.4)
(Anionic
trypsin I)
(Pretrypsinoge
n I)
1 TRY1_RAT 1 8.1 2 3 0.51 (P00762) LGEHNINVLEGDE 3
Anionic QFINAAK
trypsin-1
precursor (EC
3.4.21.4)
(Anionic
trypsin I)
(Pretrypsinoge
n I)
Group Protein Protein Percent Numbe Total Percent Description Peptide Sequence Precurs
Probabili Probabili Covera r of Numbe Share or Ion
ty ty ge Unique r of of Charge
Group Protein Protein Percent Numbe Total Percent Description Peptide Sequence Precurs
Probabili Probabili Covera r of Numbe Share or Ion
ty ty ge Unique r of of Charge
Peptid Peptid Spectru
es es m ID’s
1 VIME_HU 1 17.8 8 111 18.91 (P08670) ILLAELEQLK 2
MAN,VIME Vimentin,(Q5R
_PANTR 1W8) Vimentin
1 VIME_HU 1 17.8 8 111 18.91 (P08670) LLQDSVDFSLADAI 2
MAN,VIME Vimentin,(Q5R NTE
_PANTR 1W8) Vimentin
1 VIME_HU 1 17.8 8 111 18.91 (P08670) LLQDSVDFSLADAI 2
MAN,VIME Vimentin,(Q5R NTEFK
_PANTR 1W8) Vimentin
1 VIME_HU 1 17.8 8 111 18.91 (P08670) ALDIEIATYR 2
MAN,VIME Vimentin,(Q5R
_PANTR 1W8) Vimentin
M[147]
1 VIME_HU 1 17.8 8 111 18.91 (P08670) SSVPGVRLLQDSV 2
MAN,VIME Vimentin,(Q5R DFSLADAINTE
_PANTR 1W8) Vimentin