mass spectrometric analysis of proteome 
 samples from eukaryotic 
 cell lines , tissues or biological fluids is still hampered by the 
 vast complexity of samples and concentration differences of proteins 
 within making comparisons of proteomics and transcriptomics data difficult . 

online and off - line two - dimensional ( 2d ) or multidimensional separation 
 to reduce sample complexity is therefore still of high importance . 

the most widely used methods involve two subsequent steps , an off - line 
 fractionation step followed by ( low ph ) reversed phase liquid chromatography 
 ( rp - lc ) directly coupled to mass spectrometry .
ideally , the separation 
 in the two - dimensions is highly complementary and uses different mechanisms , 
 that is , they separate according to different properties of the analyte 
 such as size , charge and hydrophobicity .
many approaches have shown 
 decent orthogonality such as hydrophilic interaction liquid chromatography 
 ( hilic ) , electrostatic repulsion hydrophilic 
 interaction chromatography ( erlic ) , strong 
 cation exchange ( scx ) , isoelectric focusing ( such as 
 offgel ) , scx - weak - anion exchange ( wax ) 
 3d and high - ph rp . particularly , high ph rp chromatography has shown great potential 
 as it is highly resolving and partly orthogonal to low ph rp .
however , this setup requires the samples to be 
 dried down after the first dimension resulting in sample loss , unless 
 another , third dimension such as sax is added , which 
 increases the number of fractions and thus instrument time and reduces 
 robustness of the system .
furthermore , the high ph of the first rp 
 dimension is corroding most of the silica - based stationary phase , 
 leading to reproducibility issues and short column lifetime . while high - ph rp can never achieve full orthogonality to low - ph 
 rp as they both separate according to hydrophobicity , ion exchange 
 chromatography such as scx or strong anion exchange ( sax ) chromatography 
  at least theoretically  can .
but while scx has been 
 widely used , sax has been not widely considered for proteomic analysis . 

this is surprising as it was observed that the majority of tryptic 
 peptides cluster between pi of 3 and 5 , partly because many post - translational 
 modifications such as phosphorylation , pyro - glutamination or acetylation 
 decrease the pi .
this suggests that the 
 majority of the tryptic peptides in a total cell digest are likely 
 acidic , thus negatively charged and should be well separable by sax .
some groups reported studies involving sax fractionation , mostly 
 for phospho - proteomics and glycomics . on the proteome level ,
an online 
 mixed mode reversed phase anion exchange ( mm , rp - ax ) , a sax - scx mixed bed ion exchange chromatography , a sax microreactor or a sax stage - tip approach were successfully used ; however , the use of sax for large - scale proteome analyses 
 is still underdeveloped . here , we present a sax column with 
 a hyperbranched architecture , quaternary 
 ammonium ion functionality and an 
 ultralow hydrophobicity .
while the column was originally designed 
 for the separation of small organic and haloacetic acids , we show 
 its remarkable separation power for proteome research .
this column 
 exhibits high reproducibility , high capacity , robustness and very 
 high orthogonality when coupled to low ph rp , allowing the identification 
 of more than 9000 proteins from a raw264.7 macrophage cell lysate 
 on an orbitrap velos pro mass spectrometer within only one week of 
 instrument time .
we show that both hydrophilicity and the ion exchange 
 properties of the column are important for the high orthogonality 
 of this approach , which we term hydrophilic strong anion exchange 
 ( hsax ) chromatography .
the standard proteins mixture 
 consisted in bovine thyroglobulin , bovine serum albumin , chicken ovalbumin , 
 bovine beta - casein , cytochrome c from horse heart , lysozyme from chicken 
 eggs .
all proteins were purchased from sigma aldrich ( st . louis , mo ) . 

( walkerburn , scotland ) , while those for online system were purchased 
 from merck kgaa ( darmstadt , germany ) .
the mouse macrophage cell line raw264.7 was obtained from atcc and 
 grown in dmem , 10% heat - inactivated fetal bovine serum ( fbs , sigma ) , 
 2 mm l - glutamine , 5000 u / ml penicillin and 5000 g / ml 
 streptomycin ( invitrogen ) .
raw 264.7 cells were harvested , washed 
 with ice - cold pbs and lysed in 8 m urea/50 mm tris - hcl ( ph 8.0)/10 
 mm dtt plus a phosphatase inhibitor cocktail containing 1.15 mm sodium 
 molybdate , 1 mm sodium orthovanadate , 4 mm sodium tartrate dehydrate , 
 5 mm glycerophosphate ( all sigma aldrich ) and 1 mm sodium fluoride 
 ( analar normapur , west sussex , uk ) ; finally , 1 l of benzonase 
 ( merck ) was added and the lysate passed through a 26.5 g needle . the 
 solution was left to reduce for 60 min at 30 c under shaking . 

afterward , 15 mm iodoacetamide ( iaa ) was added and the sample was 
 left to alkylate for 40 min at room temperature ( rt ) in the dark . 

the iaa was then deactivated with 20 mm dtt for 40 min at rt and at 
 this point the protein concentration was assessed by rc / dc protein 
 assay according to the manufacturer s instructions ( biorad ) . 

prior to digestion , the protein mixture was diluted 8 times with 50 
 mm tris - hcl ph 8.0 to 1 m urea .
trypsin tpck ( worthington ) was 
 methylated as described previously , added 
 to the protein mixture ( 1:100 , trypsin : 
 sample ) and the digestion was performed at rt overnight .
another 1:100 
 was added the day after for 3 h followed by addition of 1% tfa to 
 stop trypsin activity .
finally , the sample was desalted by seppack 
 oasis solid phase extraction cartridges ( waters ) , dried down and stored 
 at 80 c .
the same procedure was applied to the 
 standard protein mixture 
 (  6-mix  ) .
an off - line thermo hplc ultimate 3000 system equipped with the 
 wps-3000t(b ) fc autosampler , the dgp-3600bm pump system including 
 the srd-3600 system for solvent autodegasser , the vwd-3400rs uv / vis 
 photometer and the tcc-3000sd thermo - controlled column compartment 
 were used . in this work , we compared three different strong 
 anion exchange ( sax ) columns : the as24 , the as11-hc and the as15 ( ionpac 
 series , thermo - fisher scientific ) that were chosen according to their 
 chemical / physical characteristics .
the as24 ( 2  250 mm , 
 2000  pore size ) is a low bleed 
 column primarily designed for the separation of environmental ions 
 as well as for ion chromatography separation coupled with mass spectrometry . 

as24 is a high capacity ( 140 eq ) analytical column and it is 
 compatible with ph 014 eluents and samples ( table 1 ) .
it consists in a supermacroporous resin with 
 alkanol quaternary ammonium ions as functional groups , which are ultralow 
 hydrophobic .
it uses hyperbranched chemistry with extremely hydrophilic 
 architecture whereas as11-hc and as15 
 used aromatic monomers and are thus significantly more hydrophobic .
finally , the as25 is very similar to as24 but it is 
 the most hydrophilic 
 member of the product family ( ionpac as25 , 2  250 mm , 2000  , 
 87.5 eq of capacity ) but has a lower capacity .
it was tested 
 to confirm that the hydrophilic architecture of as24 improves the 
 orthogonal separation of tryptic peptides . to compare the results 
 of sax / rp with ( high ph ) rp/ ( low ph ) rp
, 
 two reversed - phase columns were tested off - line in the first dimension , 
 which differ in matrix composition . in this work ,
acclaim 120 c18 
 ( a conventional rp , 3 m , 120  , 2.1  150 
 mm , thermo - fisher scientific ) and acclaim rslc polar advantage ii 
 -pa2- ( a polar - embedded rp , 2.2 m , 120  , 2.1  
 100 mm , thermo - fisher scientific ) columns were used to separate tryptic 
 digested proteins from raw264.7 cell lysate .
while both c18 columns 
 are based on ultrapure silica , acclaim pa2 has the advantage to have 
 specially designed amide - embedded ligands .
those features make the 
 column compatible with 100% aqueous environments over a wide range 
 of ph ( 1.510 ) , exhibiting high polarity for selectivity complementary 
 to conventional rp columns .
the acclaim pa2 column provided 
 higher efficiencies than the acclaim 
 120 c18 column ( supporting information , 
 figure s1 ) and therefore it was chosen for the comparison with the 
 as24 . for the off - line 2d lc , a highly resolving c18 column 
 was used 
 ( gemini - c18 , 3 m , 110  , 3  250 mm , phenomenex ) . 

this column uses ultrapure spherical silica particles and can operate 
 at wide ph range ( 112 ) increasing method development flexibility .
peptide separation on the off - line 1d separation system employing 
 strong anion exchange was achieved with a flow rate of 0.25 ml / min 
 ( solvent a : 20 mm tris - hcl ph 8.0 ; solvent b : 20 mm tris - hcl ph 8.0 , 
 1 m nacl ) .
a gradient ( slope 7 for 6-mix , 8 for raw264.7 cells ) spanning 
 0100% mobile phase b over 35 min was used . in case of high 
 ph rp
, separation was based on an acetonitrile ( acn ) gradient ( solvent 
 a : 20 mm nh4oh / h2o , ph 10.0 ; solvent b : 20 mm 
 nh4oh / acn , ph 10.0 ) at the same flow rate ( 0.250 ml / min , 
 080% b in 35 min ) .
when the acclaim 120 was tested , the acn 
 gradient was set at 580% b for 35 min . the off - line 
 2d
was commonly performed at ph 2.5 ( solvent a : 0.1% 
 tfa / h2o ; solvent b : 0.08% tfa / acn ) at a flow rate of 0.4 
 ml / min and a multistep gradient of 35 min : 515% b in 5 min , 
 following by 30 min up to 90% b. all chromatograms were recorded 
 by a uv detector at 214 nm . off - line 1d - fractions ( either 
 sax without further handling or high ph rp , after drying down and 
 resuspension in 0.1% tfa ) from the raw264.7 cell lysate were run online 
 on an orbitrap velos pro ( thermo - fisher scientific ) .
all nanoflow 
 chromatography was performed on an ultimate 3000 nano lc system ( thermo - fisher 
 scientific ) , using an acclaim pepmap 100 ( 75 m i d  500 
 mm , 3 m c18 ) in conjunction with an acclaim c18 pepmap trapping 
 column ( 100 m i d  20 mm , 5 m c18 ) ( thermo - fisher 
 scientific ) . linear gradient elution was performed using buffer a 
 ( 0.1% formic acid ) and buffer b ( 0.08% formic acid , 80% acn ) starting 
 from 5% buffer b to 35% over 277 min at a flow rate of 300 nl / min . 

a coated silica tip ( new objective , woburn , ma ) was used for electrospraying 
 the sample into the mass spectrometer , at an ion spray voltage of 
 1.45 kv .
ms analysis was operated in data dependent mode , such that 
 the top 20 most abundant precursors in each ms scan were subjected 
 to ms / ms ( cid in the linear trap , normalized collision energy = 35% , 
 precursor isolation width = 2 da , intensity threshold for precursor 
 selection = 2000 ) . as global parameters , precursor ions between m / z 4002000 were selected , at resolution 
 of 60000 collected in profile mode .
dynamic exclusion was enabled with a repeat count of 1 and 
 exclusion duration set to 60 s. the lock mass feature was enabled 
 for m / z = 445.120025 ( [ si(ch3)2o]6 ) as the internal calibrant ion .
the data analysis of all ms / ms proteomics 
 data sets was performed through the trans - proteomic pipeline ( tpp ) 
 as previously described .
briefly , mass 
 spectrometry raw output files were first converted to mzxml by readw and afterward searched through the x!tandem 
 ( cyclone 2011.12.01 ) search engine with k - score plug - in .
data was searched against a randomized forward 
 and reverse ipi mouse database ( version 3.87 ) and x!tandem parameters 
 included cysteine carbamylation as fixed modifications , methionine 
 oxidation and asparagine / glutamine deamidation were set as variable 
 modifications .
cyclization of glutamine / glutamic acid at the n - termini 
 ( pyroglutamate ) was automatically searched , since tandem automatically 
 checks for formation of pyroglutamic acid , that is , the loss of water 
 ( e ) or ammonia ( q ) , respectively , when a peptide starts with e or 
 q. this modification is considered to be an n - terminal modification 
 only , so it does not affect any potential modifications specified 
 for q , e or c. search parameters specified a precursor mass tolerance 
 of 25 ppm ( required by tpp , typical mass accuracy was < 2 
 ppm ) , a ms / ms tolerance at 0.4 da and full trypsin specificity allowing 
 for up to 2 missed cleavages .
peptideprophet was then used to validate the search engine results and 
 to assign accurate probabilities to peptide 
results were filtered at a calculated 1% fdr 
 on the peptide level and then protein level .
data were imported into 
 r ( v2.11.0 ) , an open source platform for statistical analysis ( http://www.r-project.org ) and graphs were plotted using the 
 ggplot2 library .
for identification 
 via andromeda , raw - files 
 were loaded into maxquant 1.3.0.5 and 
 searched against the same ipi mouse database ( version 3.87 ) .
variable 
 and fixed modifications were the same as above ; precursor mass accuracy 
 was set to 6 ppm , ms / ms tolerance to 0.4 da and trypsin specificity 
 allowing for up to 2 missed cleavages . false - discovery rate was set 
 to 0.05 for peptides and 0.01 for proteins .
from a range of 12 high - capacity 
 hydroxide - selective anion - exchange 
 columns ( ionpac series , thermo - fisher scientific ) which were initially 
 developed for the separation of small organic and haloacetic acids 
 and inorganic anions , we tested three members ( as24 , 
 as11-hc and as15 ) for the use in off - line fractionation in a proteomics 
 setting .
these columns all contain the same functionality of alkanol 
 quaternary ammonium ions , but differ in backbone hydrophobicity , with 
 as24 exhibiting ultralow hydrophobicity , as11-hc medium hydrophobicity 
 and as15 high hydrophobicity . in initial experiments we injected 
 180 g of a tryptic digest 
 of six proteins
fractions were automatically collected and reinjected onto a c18 rp 
 column using an ultimate 3000 hplc ( thermo - fisher scientific ) and 
 data acquired on a uv detector . with the use of chromeleon software 
 ( v6.8 ) ,
2d retention maps were created by which the evaluation of 
 the peptide distribution over the two dimensions was possible .
retention 
 maps showed a highly orthogonal separation by as24as shown 
 by the wide distribution over the two - dimensional space  some 
 separation by as11-hc and almost full retention of peptides on as15 
 even when eluted with 1 m nacl ( figure 1a ) . 

as the functionality in all three materials is identical , the difference 
 of separation efficiency must depend on their differences in hydrophobicity . 

indeed , addition of at least 25% acn to the mobile phase allowed elution 
 of tryptic peptides from as15 and improved elution from as11-hc ( figure 1b ) , showing that high hydrophobicity of the chemical 
 backbones of these columns hampers the use for separation 
 of peptides .
furthermore , the most hydrophilic member of the ion exchange 
 materials in this line ( as25 ) performs similarly well as as24 ( supporting information , figure s2 ) which led 
 us to conclude that both the hydrophilicity in conjunction with the 
 ion exchange functionality are important for the high orthogonality 
 of this type of chromatography that we termed hydrophilic strong anion 
 exchange ( hsax ) chromatography .
( a ) 2d retention maps of peptides of a 6-protein mix digest show that 
 elution with up to 1 m nacl in absence of organic solvent leads to 
 high orthogonality for as24 ( ultralow hydrophobicity ) , but part and 
 full retention of peptides by as11-hc ( medium hydrophobicity ) and 
 as15 ( high hydrophobicity ) , respectively .
( b ) addition of 25% acetonitrile 
 ( acn ) into the elution buffer enhances elution from as11-hc and as15 . 

( c ) three replicate injections on as24 over the course of 48 h show 
 high reproducibility .
( d ) increasing amounts of protein digest loaded 
 on as24 show good linear behavior without peak broadening .
as the functional 
 groups are primarily quaternary amines , changes of ph have no to little 
 effect on column chemistry , but will change the available negative 
 charge on peptides .
when the mobile phase was adjusted to ph 3 , a 
 major breakthrough was observed in 2d retention maps , which was absent 
 at ph 8 .
furthermore , acidic ph in sample solution , while keeping 
 the mobile phase at ph 8 , or changing the mobile phase to ph 10 reduced 
 retention of peptides or had no beneficial effect ( supporting information , figure s3 ) .
we therefore performed 
 all following experiments in 20 mm tris - hcl , at ph 8 .
this has actually 
 the benefit that tryptic digestion of the cell lysate can be performed 
 at 20 mm tris - hcl , ph 8 and directly injected on the column , avoiding 
 solid phase extraction or lyophilization and , therefore , minimizing 
 sample loss . now that running conditions were optimized , we 
 calculated the column 
 efficiency of the as24 on a digested peptide mixture from bsa ( supporting information , figure s4 ) and we analyzed 
 the run - to - run reproducibility and linearity which is required for 
 quantitative proteomics experiments , using replicate injections of 
 digested 6-mix samples over a period of 48 h ( figure 1c ) and injections of increasing material ( figure 1d ) . in both cases , as24 showed very high reproducibility 
 ( supporting information , figure s5 ) and 
 good linearity ( supporting information , 
 figure s6 ) .
after optimized parameters were established , the method was applied 
 to a very complex mixture , a total cell lysate of the mouse macrophage 
 cell line raw264.7 .
tryptic peptides of 200 g of cell lysate 
 were separated on the as24 column in a 35 min gradient from 0 to 1 
 m nacl ( 34 fractions ) which were automatically collected and injected 
 onto a gemini c18 column ( phenomenex ) .
2d retention maps show a very 
 high orthogonality of separation between the two chromatographic dimensions 
 ( figure 2a ) , displayed by the elution of peptides 
 over the entire 2d map .
separation of total cell lysate digests by hsax 
 and a high ph / low 
 ph reversed phase approach show differences in orthogonality .
tryptic 
 raw264.7 macrophage cell lysate digests were separated on an as24 
 sax column ( a ) or an acclaim pa2 rp column at ph 10 ( b ) .
thirty - four 
 fractions of both first dimensions were separated in the second dimension 
 by reversed phase chromatography at ph 3 .
furthermore , we separated the same cell lysate 
 digest on two different 
 columns using high ph ( ph 10)/ low ph ( ph 3 ) rp / rp chromatography 
 ( supporting information , figure s1 ) which 
 has shown good orthogonality in the past and is currently seen as 
 the gold standard in two - dimensional separation due to its high resolution .
we then optimized elution parameters and chose the acclaim pa2 ( thermo - fisher 
 scientific ) as it performed best , likely due to its higher polarity 
 than the conventional c18 ( acclaim 120 , thermo - fisher scientific ) . 

then we compared its orthogonality to the as24 ion exchange column 
 using 2d peptide maps using identical run time and numbers of fractions 
 ( figure 2b ) .
2d retention maps show that 
 the hsax / rp approach performs considerably 
 better than the rp / rp approach , as a much greater area in the 2d maps 
 is covered with peaks , while peptides in the rp / rp approach are scattered 
 around the diagonal .
this provides further evidence that high ph rp / low 
 ph rp can not be fully orthogonal , as both dimensions separate according 
 to hydrophobicity .
recent approaches to concatenate early and late 
 fractions of high ph rp to increase orthogonality have improved results ; however , concatenation involves user interference 
 and might provide problems in quantitative proteomics experiments 
 when the same peptides are then present in non - neighboring fractions 
 as quantitative proteomics software such as maxquant requires peptides 
 to be in neighboring fractions for accurate quantitation . in 
 order to test both hsax and high ph rp approaches for their 
 performance in a proteomics experiment , we injected aliquots ( 36% , 
 corresponding to 300400 ng of protein digest ; 1 
  10 base peak chromatogram ) of each of the 34 fractions 
 of the raw264.7 cell lysate separated by hsax or high ph rp onto an 
 orbitrap velos pro mass spectrometer , using 300 min gradients on a 
 75 m  50 cm pepmap c18 column ( thermo - fisher scientific ) , 
 corresponding to one week of instrument time each .
online separation 
 of hsax and high ph rp fractions showed a similar degree of orthogonality 
 comparable to the offline 2d chromatography ( supporting 
 information , figure s7a and b ) .
ms / ms spectra were searched 
 against the mouse ipi database v3.87 
 using x!tandem .
results were filtered at peptide- and proteinprophet 
 estimated 1% fdr . while we identified with the rp / rp approach 99110 
 peptides and 8627 proteins ( 1% fdr ) , the hsax approach resulted in 
 identification of 126318 unique peptides and 9469 proteins , representing 
 28% and 10% more identifications respectively ( table 2 and supporting information , table 
 s1 ) , while keeping the average sequence coverage equal ( 31% respectively 
 32% ) ( supporting information , figure s8 ) . 
 combining both approaches
, we identified 159847 peptides and 9871 
 proteins showing that we could increase the number of peptides and 
 proteins identified by only 20% and 4% respectively compared to hsax 
 alone ( table 2 ) .
in addition , we analyzed the 
 data using the andromeda search engine included in maxquant which identified overall 68% less proteins and peptides 
 in each experiment , but confirmed the superiority of our sax approach 
 ( table 2 and supporting 
 information , table s1 ) .
this data compares well to two recent 
 publications in which about similar number of proteins were identified 
 from a mammalian cell line using 3 different proteases and 2 weeks 
 of instrument time or in which samples 
 were analyzed repeatedly with inclusion lists .
unique proteins ( fdr  1% ) 
 according to proteinprophet ( x!tandem ) and maxquant ( andromeda ) .
unique nonstripped peptides 
 ( fdr 
  1% ) according to peptideprophet ( x!tandem ) and maxquant ( andromeda ) .
number differing from figure 4a due to fdr recalculation on combined pep.xml ( peptideprophet ) .
the high orthogonality of the hsax approach is not 
 only shown by 
 the broad distribution in the 2d peptide maps , but also exemplified 
 by the fact that we identified more than 3000 proteins in 16 out of 
 34 fractions , while the rp / rp approach achieved this for only 6 fractions 
 ( figure 3a ) .
data also indicated that we could 
 have saved instrument time by combining the last 5 fractions , which 
 could have reduced run time by almost another day .
the hsax approach 
 also showed a considerably higher number of proteins ( 7292 vs 6315 
 in rp / rp , + 15% ) that were identified with 3 and more peptides which 
 considerably improves identification and quantitation in proteomics 
 experiments .
comparison of protein and peptide identifications from 
 off - line 
 separation by either hsax / rp or rp / rp and lc  ms experiments . 
 ( a )
the high orthogonality of the hsax approach is exemplified by 
 the identification of more than 3000 proteins in 16 out of 34 fractions , 
 outperforming rp / rp approach ( 6 out of 34 fractions ) .
( b ) moreover , 
 the hsax was comparable to the rp / rp approach in separating peptides 
 in a single fraction ( 69% of rp peptides were present in one fraction 
 compared to 55% for the hsax approach ) , which is beneficial for quantitative 
 proteomics experiments .
as quantitative proteomics performs best when peptides 
 are only 
 present in one fraction , we tested how many peptides were only identified 
 in a single fraction . here , the rp / rp approach expectedly outperformed 
 the hsax approach , showing a higher resolution as 69% of peptides 
 were present in one fraction compared to 55% for the hsax approach 
 ( figure 3b ) .
only 18.5% ( hsax ) and 11.6% ( rp ) 
 of all peptides respectively were identified in 3 or more fractions . 

in addition , separate experiments with dimethyl - labeled proteome samples indicated that deuterated samples 
 do not change retention time in hsax chromatography as they can do 
 in rp experiments ( data not shown ) , which can potentially separate 
 light and heavy forms of isotopically labeled peptides into neighboring 
 fractions complicating peptide quantitation .
interestingly , 
 the two techniques seem to favor certain subsets 
 of peptides as only 58679 peptides ( 46% for hsax and 60% for rp ) are 
 shared among the two approaches ( figure 4a ) . to further identify the characteristics of these 
 subsets
, we analyzed which amino acids were over - represented in the 
 data sets unique to each approach ( supporting 
 information , figure s9 ) and identified peptides with a higher 
 number of acidic residues to be the major contributor to the difference 
 ( figure 4b ) .
furthermore , the peptides identified 
 by the hsax approach matched much better to an in silico digest of all proteins of the mouse ipi database ( figure 4b ) , suggesting that the hsax approach allows for 
 the identification of more representative peptides of the total proteome 
 than the rp / rp approach .
hsax / rp and rp / rp lead to the identification 
 of different peptide 
 subsets .
( a ) both hsax and rp approaches lead to the identification 
 of different subsets of peptides with only about half the peptides 
 identified in the two approaches ( please note : number of combined 
 peptides higher than in table 2 due to fdr 
 recalculation ) ( proportional venn diagram has been produced using 
 bioinforx ) .
( b ) in - depth analysis of the amino acid distribution in 
 the subset of unique peptides revealed that hsax favored more acidic 
 peptides than rp , a trend that is more representative to the in - silico tryptic digestion of the whole ipi database .
( c ) 
 moreover , both hsax and rp approaches showed similar peptide charge 
 state distribution over the whole gradient , favoring doubly and triply 
 charged peptide ions .
finally , we tested how separation via hsax affected 
 the charge - state 
 distribution of peptide ions in mass spectrometry over the gradient . 

unlike scx , peptides fractionated by sax should not show a charge - distribution 
 as the negative charges by which the peptides were separated in the 
 first dimension at ph 8 do not affect electrospray ionization ( esi ) 
 after separation at ph 3 in the second dimension , where basic residues 
 are critical .
evaluation of peptide charge states showed that peptides 
 separated by both hsax and rp / rp showed a relatively even charge state 
 distribution over the whole gradient ( figure 4c ) .
this is of great advantage as the identification of doubly and 
 triply charged peptide ions is highly favored in esi 
ms / ms 
 compared to singly and highly charged ions which populate , for example , 
 early and late fractions of scx experiments .
in this work we demonstrate that hydrophilic 
 strong anion exchange 
 chromatography provides a robust and reproducible separation of complex 
 protein digest samples for proteomics analysis .
its very high orthogonality 
 proved to be superior to an optimized high ph rp / low ph rp approach 
 and enabled us to identify > 9,000 proteins from raw264.7 mouse 
 macrophage 
 cell lysate in just one week of mass spectrometry instrument time 
 which will allow a better comparison of proteomics and transcriptomics 
 data . in the future , we will test the applicability of the hsax material 
 for online 2d lc - ms and the separation of complex mammalian proteomes 
 for quantitative proteomics as well as phosphopeptide analysis .
 due to its compatibility and orthogonality to reversed 
 phase ( rp ) 
 liquid chromatography ( lc ) separation , ion exchange chromatography , 
 and mainly strong cation exchange ( scx ) , has often been the first 
 choice in multidimensional lc experiments in proteomics . here , we 
 have tested the ability of three strong anion exchanger ( sax ) columns 
 differing in their hydrophobicity to fractionate raw264.7 macrophage 
 cell lysate . 
 ionpac as24 , a strong anion exchange material with ultralow 
 hydrophobicity , demonstrated to be superior to other materials by 
 fractionation and separation of tryptic peptides from both a mixture 
 of 6 proteins as well as mouse cell lysate . 
 the chromatography displayed 
 very high orthogonality and high robustness depending on the hydrophilicity 
 of column chemistry , which we termed hydrophilic strong anion exchange 
 ( hsax ) . 
 mass spectrometry analysis of 34 sax fractions from raw264.7 
 macrophage cell lysate digest resulted in an identification of 9469 
 unique proteins and 126318 distinct peptides in one week of instrument 
 time . 
 moreover , when compared to an optimized high ph / low ph rp separation 
 approach , the method presented here raised the identification of proteins 
 and peptides by 10 and 28% , respectively . 
 this novel hsax approach 
 provides robust , reproducible , and highly orthogonal separation of 
 complex protein digest samples for deep coverage proteome analysis . 
Introduction
Experimental Section
Results and Discussion
Conclusion