multiple myeloma ( mm ) is a clonal b - cell disorder in which malignant plasma cells ( pcs ) accumulate in the bone marrow , resulting in lytic bone lesions and excessive amounts of monoclonal proteins .
although therapeutic interventions have been developed , and the overall survival has been improved over the last decade , almost all patients with mm , who survive initial treatment will develop drug resistance and relapse .
the potential therapeutic targets in human mm are investigated by differentially profiling of purified mm and normal pcs using two - dimensional ( 2d ) gel electrophoresis .
a total of 43 differentially expressed proteins are identified and six of them are confirmed with western blotting , including profilin-1 , glutathione peroxidase 1 , annexin a1 , proteasome subunit alpha type-5 , proteasome activator subunit-2 , and proteasome subunit beta type-10 .
extensive proteomic studies have also been carried out for studying the response of myeloma cells to drug treatment .
however , few studies have reported the identification of markers for mm diagnosis . a recent study by rajpal et al . identified a biomarker panel from serum that predicts the response to thalidomide - based therapy in newly diagnosed myeloma patients . by using a combination of immunodepletion and 2d - dige ,
differentially expressed proteins between responders and nonresponders were detected including zinc - a2-glycoprotein , vitamin d - binding protein , serum amyloid a ( saa ) , 2-microglobulin ( bmg ) , and haptoglobin developed a method for assessing the quality of surface - enhanced laser desorption / ionization time - of - flight ( seldi - tof ) data based on a correlation matrix .
lytic of bone disease study to efficiently identify low - quality spectra prior to postanalysis .
the results demonstrate the potential application of this method in the identification of biomarkers for myeloma associated lytic bone disease .
recently , we applied the seldi - tof ms and matrix - assisted laser desorption / ionization tof ( maldi - tof ) ms to identify serum biomarkers of mm
. the limitations of seldi - tof analysis include low resolution and the intrinsic limitation of maldi - tof as a quantitative tool .
furthermore , it is difficult to further identify proteins on chips or weak cation exchange beads .
therefore , the combination of protein fractionation and liquid chromatography - tandem mass spectrometry ( lc - ms / ms ) with high - resolution mass spectrometer is an alternative method for protein profiling and biomarker discovery .
one challenge to identify these candidate biomarkers from serum or plasma is that there is an extensive dynamic concentration range of proteins in bio - fluids .
serum albumin is the major carrier and transporter at a concentration of about 45 mg / ml . however , most of the important indicators changing in physiological states may be present at < 1 pg / ml . methods have been developed to deplete the high abundance proteins derived from serum / plasma , such as immunodepletion , organic precipitation , affinity purification and solid phase extraction .
peptide ligand library affinity chromatography is a novel method for capturing and identifying the low abundance proteins . in this method ,
a solid - phase combinatorial library of hexapeptides is coupled , via a short spacer , on poly ( hydroxymethecrylate ) beads , by a modified merrifield approach .
the hexapeptide ligands are synthesized from natural amino acids , so the library contains millions of linear hexapeptide that are able to interact with a very large number of proteins present in a complex proteome .
have reported a large - scale proteomic study of human serum using peptide library beads and mass spectrometry .
analysis of the elution from this combinatorial library resulted in the identification of 1559 proteins including a large number of low abundance proteins . in the present work
, we applied the peptide ligand library beads ( pllbs)-based affinity method to the differential analysis of myeloma serum samples versus serum samples of healthy individuals . using a label - free semi - quantitative approach
, we found that expression levels of 22 proteins significantly differed between serum samples from healthy control and mm patients .
all the patients with mm from the mm research center at the beijing chao - yang hospital ( beijing , china ) were diagnosed according to the durie - salmon staging system .
the healthy controls were obtained from the medical examination center of the beijing chao - yang hospital .
the research protocol was approved by the ethics committee of beijing chao - yang hospital .
serum samples were obtained from the peripheral blood by centrifuging at 4000 r / min for 10 min at 4c , and stored at 80c until analyzed .
a 300 l sample of the pooled serum samples was centrifuged to eliminate particles in suspension .
eight milligram of the pllb resin ( peptide international , lexington , ky ) was suspended in 100 l of 50% methanol for 10 min and was washed three times with phosphate - buffered saline ( pbs ) solution ( ph = 7.4 ) .
the pooled serum samples were incubated with the pllb resin at room temperature ( 2225c ) on a soft shaker for 2 h. after removing the unbound fraction , the pllb resin was washed three times with pbs solution .
proteins were eluted from the beads by incubating with lds sample buffer ( invitrogen , grand island , ny , usa ) at 100c for 5 min .
proteins were separated on a 412% gradient tris - glycine sodium dodecyl sulfate ( sds)-gel ( invitrogen , usa ) , and the gel bands were stained with colloidal coomassie blue ( invitrogen ) .
each lane was cut into 15 slices and each gel slice was reduced with 10 mmol / l dtt and alkylated with 100 mmol / l iodoacetamide .
then in - gel digestion was carried out with the sequence grade modified trypsin ( promega ) in 50 mmol / l ammonium bicarbonate at 37c overnight .
the peptides were extracted twice with 1% trifluoroacetic acid in 50% acetonitrile aqueous solution for 30 min . for
lc - ms / ms analysis , each digestion product was separated by a 60 min gradient elution at a flow rate of 0.25 l / min with the dionex 3000 nano - hplc system , which was directly interfaced with the thermo ltq - orbitrap mass spectrometer .
the analytical column was a home - made fused silica capillary column ( 75 m i d , 100 mm length ; upchurch , usa ) packed with c-18 resin ( 300 a , 5 m , varian , usa ) .
mobile phase a consisted of 0.1% formic acid , and mobile phase b consisted of 100% acetonitrile and 0.1% formic acid .
the ltq - orbitrap mass spectrometer was operated in the data - dependent acquisition mode using the xcalibur 2.0.7 software ( thermo fisher scientific inc . ,
usa ) and there was a single full - scan mass spectrum in the obritrap ( 4001800 m / z , 30,000 resolution ) , followed by 6 data - dependent ms / ms scans in the ion trap at 35% normalized collision energy .
the ms / ms spectra from each lc - ms / ms run were searched against ipi human database by sequest in proteindiscover .
the following search parameters were used in all of the sequest searches : maximum of 2 missed trypsin cleavages , cysteine carbamidomethylation as a fixed modification , methionine oxidation and n / q deamination as the variable modification .
the maximum error tolerance was 20 ppm for ms and 0.8 da for ms / ms .
the filter is score 1.9 versus 1 charges , 2.2 versus 2 charges , 2.75 versus 3 charges , and at least 1 peptide every protein .
quantitation of protein expression by spectra counts for each identified proteins was carried out using an in - house developed perl script .
significance was regarded only when the ratio of spectra counts between two groups were more than 2 or < 0.5 .
extracted ion currents for selected peptides were also used to quantify the protein concentrations from different samples .
sixty microgram of protein from the pooled serum of 8 patients with mm and eight healthy controls was separated in nupage novex 412% bis - tris gradient gels ( invitrogen ) and transferred to a nitrocellulose membranes ( millipore , billerica , ma , usa ) for 30 min using 1  nupage transfer buffer .
nitrocellulose membranes were blocked overnight at 4c in blocking buffer ( 5% skim milk , 0.1% tween 20 , 0.01 mol / l pbs ) .
after washes with washing buffer ( 0.1% tween , 0.01 mol / l pbs ) , the membranes were incubated with primary antibodies in the blocking buffer for 120 min .
antiantibodies antibodies made against specific to integrin alpha-1 and isoform-1 of multimerin-1 ( abcam , cambridge , ma , usa ) were used at 1:1000 and 1:2000 dilution , respectively . after washing ,
the membrane was incubated with second antibodies ( goat anti - mouse / hrp conjugate , santa cruz , paso robles , ca , usa ) for integrin alpha-1 and isoform-1 of multimerin-1 and detected with the pierce chemiluminescent ( ecl ) kit ( thermo , waltham , ma , usa ) . levels of proteins were then quantified by image quant tl activation wizard software , usa .
all the patients with mm from the mm research center at the beijing chao - yang hospital ( beijing , china ) were diagnosed according to the durie - salmon staging system .
the healthy controls were obtained from the medical examination center of the beijing chao - yang hospital .
the research protocol was approved by the ethics committee of beijing chao - yang hospital .
serum samples were obtained from the peripheral blood by centrifuging at 4000 r / min for 10 min at 4c , and stored at 80c until analyzed .
a 300 l sample of the pooled serum samples was centrifuged to eliminate particles in suspension .
eight milligram of the pllb resin ( peptide international , lexington , ky ) was suspended in 100 l of 50% methanol for 10 min and was washed three times with phosphate - buffered saline ( pbs ) solution ( ph = 7.4 ) .
the pooled serum samples were incubated with the pllb resin at room temperature ( 2225c ) on a soft shaker for 2 h. after removing the unbound fraction , the pllb resin was washed three times with pbs solution .
proteins were eluted from the beads by incubating with lds sample buffer ( invitrogen , grand island , ny , usa ) at 100c for 5 min .
proteins were separated on a 412% gradient tris - glycine sodium dodecyl sulfate ( sds)-gel ( invitrogen , usa ) , and the gel bands were stained with colloidal coomassie blue ( invitrogen ) .
each lane was cut into 15 slices and each gel slice was reduced with 10 mmol / l dtt and alkylated with 100 mmol / l iodoacetamide .
then in - gel digestion was carried out with the sequence grade modified trypsin ( promega ) in 50 mmol / l ammonium bicarbonate at 37c overnight .
the peptides were extracted twice with 1% trifluoroacetic acid in 50% acetonitrile aqueous solution for 30 min . for lc - ms / ms analysis ,
each digestion product was separated by a 60 min gradient elution at a flow rate of 0.25 l / min with the dionex 3000 nano - hplc system , which was directly interfaced with the thermo ltq - orbitrap mass spectrometer .
the analytical column was a home - made fused silica capillary column ( 75 m i d , 100 mm length ; upchurch , usa ) packed with c-18 resin ( 300 a , 5 m , varian , usa ) .
mobile phase a consisted of 0.1% formic acid , and mobile phase b consisted of 100% acetonitrile and 0.1% formic acid .
the ltq - orbitrap mass spectrometer was operated in the data - dependent acquisition mode using the xcalibur 2.0.7 software ( thermo fisher scientific inc . ,
usa ) and there was a single full - scan mass spectrum in the obritrap ( 4001800 m / z , 30,000 resolution ) , followed by 6 data - dependent ms / ms scans in the ion trap at 35% normalized collision energy .
the ms / ms spectra from each lc - ms / ms run were searched against ipi human database by sequest in proteindiscover .
the following search parameters were used in all of the sequest searches : maximum of 2 missed trypsin cleavages , cysteine carbamidomethylation as a fixed modification , methionine oxidation and n / q deamination as the variable modification .
the maximum error tolerance was 20 ppm for ms and 0.8 da for ms / ms .
the filter is score 1.9 versus 1 charges , 2.2 versus 2 charges , 2.75 versus 3 charges , and at least 1 peptide every protein .
quantitation of protein expression by spectra counts for each identified proteins was carried out using an in - house developed perl script .
significance was regarded only when the ratio of spectra counts between two groups were more than 2 or < 0.5 .
extracted ion currents for selected peptides were also used to quantify the protein concentrations from different samples .
sixty microgram of protein from the pooled serum of 8 patients with mm and eight healthy controls was separated in nupage novex 412% bis - tris gradient gels ( invitrogen ) and transferred to a nitrocellulose membranes ( millipore , billerica , ma , usa ) for 30 min using 1  nupage transfer buffer .
nitrocellulose membranes were blocked overnight at 4c in blocking buffer ( 5% skim milk , 0.1% tween 20 , 0.01 mol / l pbs ) .
after washes with washing buffer ( 0.1% tween , 0.01 mol / l pbs ) , the membranes were incubated with primary antibodies in the blocking buffer for 120 min .
antiantibodies antibodies made against specific to integrin alpha-1 and isoform-1 of multimerin-1 ( abcam , cambridge , ma , usa ) were used at 1:1000 and 1:2000 dilution , respectively .
after washing , the membrane was incubated with second antibodies ( goat anti - mouse / hrp conjugate , santa cruz , paso robles , ca , usa ) for integrin alpha-1 and isoform-1 of multimerin-1 and detected with the pierce chemiluminescent ( ecl ) kit ( thermo , waltham , ma , usa ) . levels of proteins were then quantified by image quant tl activation wizard software , usa .
the patients in our study were newly diagnosed with mm and their serum samples were collected prior to any treatment .
bmg is a prognostic factor , and albumin is relevant to the patients health status .
concentrations of bmg and serum albumin for mm patients bmg : 2-microglobulin ; mm : multiple myeloma . as shown in figure 1 , separation of 2 l of pooled serum
is displayed in lanes 1 and 2 , indicating that major proteins that is albumin , are overwhelming and make low abundance proteins less visible on the gel .
two elutaions from pllbs are shown in lanes 3 and 4 , indicating the enrichment of low - abundance proteins and the drastically reduced levels of the most abundant proteins present in the serum .
lane 1 and lane 2 : 2 l of the pooled serum ; lane 3 and lane 4 : the elutions from the peptide ligand library beads .
identification of proteins by 1d - gel - lc - ms / ms : gel bands were excised and digested with trypsin .
the tryptic peptides from each gel band were analyzed by lc - ms / ms .
based on the searching criteria for protein hits , 849 and 789 proteins were identified from samples of healthy controls and patients with mm , respectively . the false positive rate for proteins identified with mascot , as calculated by the decoy database search ,
although the number of proteins identified is not as high as what has been reported , our results are more reliable since the ms measurement was carried out with a ltq - obritrap mass spectrometer with the mass measurement error less than 10 ppm .
the studies carried out with ltq usually use 3 da as the mass measurement error , which greatly increases the false positive rate . using pllb treatment
, we were able to identify some low abundance proteins and proteins that are seldom implicated in mm , such as integrin alpha-11 and isoform-1 of multimerin-1 .
the ms / ms spectra of selected tryptic peptides fvlvqenrptltdivelr from isoform-1 of multimerin-1 and iqnlglfpihgmmmk from integrin alpha-11 are displayed in figure 2a and b , respectively , in which all major fragment ions match the b and y ions of the peptides from these two proteins .
( a ) the mass spectrometry ( ms)/ms spectra of selected tryptic peptides fvlvqenrptltdivelr from isoform-1 of multimerin-1 ; ( b ) the ms / ms spectra of selected tryptic peptides iqnlglfpihgmmmk from integrin alpha-11 .
spectral count was used to quantify the expression levels of proteins in the selected gel bands , as well as the whole lane .
spectra count uses the number of spectra that has been assigned to a specific protein , to quantify the relative abundance of protein from two samples , and it has been widely applied to biological systems . using two - fold or more changes as a determinant , we identified the expression of 10 proteins down - regulated and that of 12 proteins up - regulated in patients with mm compared to healthy controls .
we also used an alternative approach , the extracted ion current , to confirm the quantitation results from the spectra counts . these up- and
differentially expressed serum proteins between patients with mm and healthy controls by pllb affinity combining with one - dimensional sds - page gel - lc - ms / ms mm : multiple myeloma ; pllb : peptide ligand library beads ; ms : mass spectrometry ; lc : liquid chromatography ; sds - page : sodium dodecyl sulphate - polyacrylamide gel electrophoresis .
two of the differentially expressed proteins were selected for immunodetection in undepleted plasma using specific antibodies to integrin alpha-11 and isoform-1 of multimerin-1 and the differential serum levels were confirmed by western blotting analysis [ figure 3 ] .
the immunoblotting analysis showed decreased integrin alpha-11 in the serum of mm patients with a mean band intensity of ( 2425  562 ) ( mean optical density units ) versus controls at ( 1177  560 ) ( p < 0.01 , student 's t - test , two - tailed ) .
isoform-1 of multimerin-1 appeared to be down - regulated in mm patients at ( 3019  641 ) versus controls at ( 11,988  846 ) p < 0.01 ) .
results from western blotting were consistent with those from spectral counts , showing that both proteins were down - regulated in the mm patients ( integrin alpha-11 , 116 spectral counts and isoform-1 of multimerin-1 , 13 spectral counts ) versus healthy controls ( integrin alpha-11 , 384 spectral counts and isoform-1 of multimerin-1 , 44 spectral counts ) .
the relative abundance profiles , as determined by western blotting analysis , were therefore consistent with the quantitative spectral counts data obtained for these two proteins .
lane 1 , lane 2 and lane 3 were the three healthy people 's serum ; lane 4 , lane 5 and lane 6 were the three patient 's serum .
the patients in our study were newly diagnosed with mm and their serum samples were collected prior to any treatment .
bmg is a prognostic factor , and albumin is relevant to the patients health status .
concentrations of bmg and serum albumin for mm patients bmg : 2-microglobulin ; mm : multiple myeloma .
as shown in figure 1 , separation of 2 l of pooled serum is displayed in lanes 1 and 2 , indicating that major proteins that is albumin , are overwhelming and make low abundance proteins less visible on the gel .
two elutaions from pllbs are shown in lanes 3 and 4 , indicating the enrichment of low - abundance proteins and the drastically reduced levels of the most abundant proteins present in the serum .
lane 1 and lane 2 : 2 l of the pooled serum ; lane 3 and lane 4 : the elutions from the peptide ligand library beads .
identification of proteins by 1d - gel - lc - ms / ms : gel bands were excised and digested with trypsin .
the tryptic peptides from each gel band were analyzed by lc - ms / ms .
based on the searching criteria for protein hits , 849 and 789 proteins were identified from samples of healthy controls and patients with mm , respectively . the false positive rate for proteins identified with mascot , as calculated by the decoy database search ,
was estimated to be 2% . although the number of proteins identified is not as high as what has been reported , our results are more reliable since the ms measurement was carried out with a ltq - obritrap mass spectrometer with the mass measurement error less than 10 ppm .
the studies carried out with ltq usually use 3 da as the mass measurement error , which greatly increases the false positive rate . using pllb treatment
, we were able to identify some low abundance proteins and proteins that are seldom implicated in mm , such as integrin alpha-11 and isoform-1 of multimerin-1 .
the ms / ms spectra of selected tryptic peptides fvlvqenrptltdivelr from isoform-1 of multimerin-1 and iqnlglfpihgmmmk from integrin alpha-11 are displayed in figure 2a and b , respectively , in which all major fragment ions match the b and y ions of the peptides from these two proteins .
( a ) the mass spectrometry ( ms)/ms spectra of selected tryptic peptides fvlvqenrptltdivelr from isoform-1 of multimerin-1 ; ( b ) the ms / ms spectra of selected tryptic peptides iqnlglfpihgmmmk from integrin alpha-11 .
spectral count was used to quantify the expression levels of proteins in the selected gel bands , as well as the whole lane .
spectra count uses the number of spectra that has been assigned to a specific protein , to quantify the relative abundance of protein from two samples , and it has been widely applied to biological systems . using two - fold or more changes as a determinant , we identified the expression of 10 proteins down - regulated and that of 12 proteins up - regulated in patients with mm compared to healthy controls .
we also used an alternative approach , the extracted ion current , to confirm the quantitation results from the spectra counts . these up- and
differentially expressed serum proteins between patients with mm and healthy controls by pllb affinity combining with one - dimensional sds - page gel - lc - ms / ms mm : multiple myeloma ; pllb : peptide ligand library beads ; ms : mass spectrometry ; lc : liquid chromatography ; sds - page : sodium dodecyl sulphate - polyacrylamide gel electrophoresis .
two of the differentially expressed proteins were selected for immunodetection in undepleted plasma using specific antibodies to integrin alpha-11 and isoform-1 of multimerin-1 and the differential serum levels were confirmed by western blotting analysis [ figure 3 ] .
the immunoblotting analysis showed decreased integrin alpha-11 in the serum of mm patients with a mean band intensity of ( 2425  562 ) ( mean optical density units ) versus controls at ( 1177  560 ) ( p < 0.01 , student 's t - test , two - tailed ) .
isoform-1 of multimerin-1 appeared to be down - regulated in mm patients at ( 3019  641 ) versus controls at ( 11,988  846 ) p < 0.01 ) .
results from western blotting were consistent with those from spectral counts , showing that both proteins were down - regulated in the mm patients ( integrin alpha-11 , 116 spectral counts and isoform-1 of multimerin-1 , 13 spectral counts ) versus healthy controls ( integrin alpha-11 , 384 spectral counts and isoform-1 of multimerin-1 , 44 spectral counts ) .
the relative abundance profiles , as determined by western blotting analysis , were therefore consistent with the quantitative spectral counts data obtained for these two proteins .
lane 1 , lane 2 and lane 3 were the three healthy people 's serum ; lane 4 , lane 5 and lane 6 were the three patient 's serum .
in the present study , we employed a novel method of peptide ligand library affinity chromatography combining with 1d - gel - lc - ms / ms analysis to identify proteins differentially expressed in the sera of mm patients compared to those of healthy controls .
it has been amply documented that the pllbs are an efficient means to reduce high abundance proteins for the identification of low - abundance proteins .
analysis of serum samples treated with pllb in triplicate resulted in the identical 1d - sds images in our experiments and in the literature .
of the 22 proteins differently expressed in our series , the expression level of apolipoprotein a - i is lower in the sera of mm patients than in sera of healthy individuals .
apolipoprotein a - i is the major apolipoprotein of high - density lipoprotein , and it promotes efflux of cholesterol from cells .
it has been found that expression of apolipoprotein a - i is decreased in ovarian cancer .
the expression of insulin - like growth factor - binding protein 2 precursor , that regulates the function of insulin is higher in the mm patients .
it has been reported that insulin - like growth factor - binding protein 2 is elevated in non - hodgkin 's lymphoma .
a recent study indicates that the expression level of saa is higher in serum from mm patients who were nonresponders to thalidomide - based therapy .
vitamin d - binding protein , that is a member of a multigene family including alpha - fetoprotein and albumin , reversibly binds and transports vitamin d and its metabolites to target cells .
our study shows that the expression of vitamin d - binding protein is decreased in mm patients .
plasma kallikrein participates in the surface - dependent activation of blood coagulation , fibrinolysis , kinin generation , and inflammation .
the expression level of plasma kallikrein was found to be elevated in mm patients in the present study .
we also found two novel proteins , integrin alpha-11 subunit and isoform-1 of multimerin-1 integrin alpha-11 was found to be overexpressed in nonsmall cell lung carcinoma ( nsclc ) and in esophageal squamous cell carcinoma .
the integrin alpha-11 subunit binds the integrin beta-1 subunit to form a heterodimer , and 11 1 integrin is one of the four receptors for interstitial collagen to modulate the growth of nsclc cells .
the expression of the integrin alpha-11 subunit is lower in mm patients compare to the normal individuals .
normally , multimerin-1 is not detectable in plasma . using both proteomic analysis and western blot
, we found that the expression of isoform-1 of multimerin-1 is decreased in mm patients .
it would be useful to compare the changes in protein concentration before and after therapy , however , we currently do not have enough data for this evaluation .
our results demonstrate that pllb affinity chromatography combining with 1d - gel - lc - ms / ms analysis is a useful method to identify proteins differently expressed in the sera of mm patients .
the changes of protein expression are mainly associated with the complement and coagulation cascade , lipid metabolism , and extracellular matrix remodeling , which concomitantly take part in the pathogenesis of mm .
 background : multiple myeloma ( mm ) is a malignant tumor , which takes the second place in malignant blood disease . 
 the clinical symptoms are complicated that make more difficult to diagnose and therapy . 
 lots of researches focus on the proteins about mm in order to solve those problems . 
 we used proteomic methods to find potential biomarkers in mm patients.methods:we applied the peptide ligand library beads ( pllbs ) to deplete high abundance proteins in serum for finding potential pathogenic factors and biomarkers of mm . using 1d - gel - liquid chromatography - tandem mass spectrometry ( lc - ms / ms ) 
 , we identified 789 and 849 unique serum proteins in mm patients and in healthy controls , respectively.results:twenty-two proteins were found differentially expressed between the two groups including serum amyloid a protein , vitamin d - binding protein isoform-1 precursor , plasma kallikrein , and apolipoprotein a - i . 
 changes of integrin alpha-11 and isoform-1 of multimerin-1 were validated with western blotting . 
 the linkage of the differentially expressed proteins and the pathogenesis pathways of mm were discussed.conclusions:pllb combined with 1d - gel - lc - ms / ms analysis is an efficient method to identify differentially expressed proteins in serum from patients with mm . 
I
M
Sample collection
Sample preparation
Protein separation on 1D-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-gel digestion and liquid chromatography-tandem mass spectrometry analysis
Data processing
Western blotting analysis
R
Subject characteristics
Peptide ligand library bead-based serum sample preparation
Differential analysis of protein expression between multiple myeloma and controls
Validation of proteomic results by Western blotting analysis
D