human immunodeficiency virus type 1 ( hiv-1 ) , the causative agent of aids , uses cd4 t cells as a host . in order to do so efficiently
the host cell , in turn , initiates intracellular antiviral responses and signals to the host s immune system ( lever and jeang , 2011 ) .
thus , hiv-1 infection and the host response trigger many physiological changes in the infected cell ( gomez and hope , 2005 ) .
hiv-1 survives and persists in infected cells preparing them for production and release of new viral particles .
intracellular changes due to hiv-1 infection have been studied extensively , focusing on the contribution of hiv-1 s accessory proteins to these processes , using microarrays or serial analysis of gene expression ( sage ) to detect mrna changes in the cell ( vant wout et al . , 2003 ; giri et al .
, 2006 ; roeth and collins , 2006 ; lefebvre et al . , 2011 ; wu et al . ,
2011 ) . gene expression profiling with microarrays is of course easy to perform , generating large datasets quickly ( heller , 2002 ) , but sequences must be known in advance , which sage does not require .
sage , based on direct sequencing of mrna tags , also does not use hybridization as microarrays do , leading to more reliable probing of mrna levels .
sage is currently being replaced by high - throughput sequencing technologies ( rna - seq ; baginsky et al . , 2010 ) .
proteome changes upon hiv infection have also been studied in detail with mass spectrometry ( coiras et al . , 2006 ; chan et al .
navare et al . , 2012 ) , lately focusing on studies specifically monitoring direct interactions between viral and cellular proteins ( jager et al .
however , changes in mrna levels are only part of the story . often stringent correlation between mrna and protein levels is lacking ( pradet - balade et al . , 2001 ) .
in human cells , transcription seems to explain only 30% of variation in protein levels , with translation and protein degradation contributing up to 40% ( vogel et al .
, 2010 ; schwanhausser et al . , 2011 ) . in e. coli , relative contributions to regulation of protein levels via transcriptional and/or translational control have even been shown to vary greatly with the kind of signal the cell responds to ( kramer et al . , 2010 ) .
a protein can exist in many different isoforms , each with its own specific function , with a relatively limited number of genes giving rise to vast amounts of ( functionally ) distinct proteins ( jensen , 2006 ) .
ptms constitute highly versatile systems allowing cells to respond very quickly to both external and internal signals , as illustrated by protein phosphorylation in signal transduction or metabolic regulation .
of course , such ptm responses can not be detected using dna / rna sequencing technologies .
thus , proteomic studies using mass spectrometry to detect and quantify differences in protein expression , protein isoforms and complexes , as well as ptms , are essential for understanding the complete set of intracellular responses to hiv-1 infection . in this way new insights and intervention strategies can be developed . in a previous study
, we used the fluorescence two - dimensional differential gel electrophoresis ( 2d - dige ) technique for a comparison of uninfected and hiv-1 infected t cells ( ringrose et al . , 2008 ) .
this technique starts out with minimal protein labeling using cyanine based fluorescent probes recognizing lysine .
a subsequent two - dimensional gel electrophoresis allows the quantification of changes in protein expression by mixing cell extracts labeled either with cy3 or cy5 and running them on a single gel ( unlu et al . , 1997 ; alban et al . , 2003 )
next , differentially expressed proteins can be identified by peptide mass fingerprinting ( pmf ) using a matrix assisted laser - desorption time of flight ( maldi - tof ) mass spectrometer .
pmf uses lists of masses of peptides (  fingerprints  ) generated by tryptic digestion of proteins for their identification .
nb : in this approach quantification is based on amount of fluorescence and not on ion detection level in a mass spectrometer .
the study confirmed several hiv-1 effects on pathways and cellular processes previously described using stable isotope labeling combined with liquid chromatography  mass spectrometry ( lc  ms ; chan et al . , 2007 ) .
but there were novel findings as well , most importantly the downregulation of proteins involved in glycolysis upon full - blown hiv-1 infection , presumably part of a complete metabolic rerouting to preserve glucose for the pentose phosphate pathway , the source of riboses for subsequent viral nucleic acid synthesis ( ringrose et al . , 2008 )
however , despite the success of this 2d - dige pmf approach it also comes with some limitations : the technique is very labor intensive and about two - thirds of all the differentially expressed proteins detected could not be identified using pmf because they were not present in sufficient abundance . as we are planning to extend our proteomic analysis of hiv-1 t cell interaction to subcellular fractions ,
we compared the 2d - dige pmf with one - dimensional separation of peptides using reversed phase lc coupled to ms ( geromanos et al .
, 2009 ) analysis , again using t cells infected with hiv-1 . in this approach target proteins
are digested with trypsin ( as in the pmf method mentioned above ) , and resulting peptides ( parent ions in figure 1 ) are now identified as coming from certain proteins by the mass analysis of their fragments ( daughter ions in figure 1 ) , which allows peptide sequencing [ in both data - dependent modes of acquisition ( dda ) and ms applications described below ] as well as protein quantitation by peptide signal abundance .  
separation of peptides ( colored dots ) on an lc coupled to a qtof instrument in a data - independent mode of acquisition using electrospray ionization ( a ) . in a data - independent mode of acquisition
the quadrupole ( ms1 ) continuously allows passage of all ions , in contrast to dda where the quadrupole selects ions for fragmentation based on their intensity .
energy in the collision cell is continuously cycled between a low and an elevated profile .
this generates spectra of all parent ( b ) and daughter ions ( d ) throughout the lc 
ms run in the time of flight analyzer ( ms2 ) without bias with respect to their relative intensities . in order to reconstruct fragment ion spectra with daughter ions from a single parent only , an ion - accounting algorithm compares the retention time profiles of all individual parent ions ( c ) to all individual daughter ions ( e ) matching them on the basis of retention time profile and intensity ( g ) . in this manner
the algorithm creates a reconstructed daughter ion spectrum matched to a single precursor ( f ) that can be used by proteome search engines to identify peptides and link these to proteins .
the unbiased data - independent scanning mode greatly expands the number of peptides  and thus of proteins  detected , compared to the dda mode of acquisition on qtof type instruments , especially using limited lc separations on complex mixtures to increase throughput .
one of the most exciting new developments in proteomic analyses is the possibility to perform quantitative protein comparisons without having to introduce quantifiable labels : label - free proteomics .
here we report on the use of label - free proteomics in a pilot study of t cells ( pm1 t cell line ) infected with hiv-1 ( lai isolate ) . in this setup a novel data - independent alternate scanning technique ( ms ) on a quadrupole time of flight ( qtof ) instrument is used .
in contrast to dda , making up the standard method on various types of instruments used in peptide based proteomics , ms does not select a single precursor ion for fragmentation but rather fragments  all  ions present at any given time during chromatographic separation .
as such , mass spectrometric data are collected ( in principle ) on fragments of  all  ions instead of a subset that is selected for fragmentation during dda analysis .
this decreases bias toward selecting only highly abundant peptides and eliminates the need to measure samples multiple times in order to collect tandem - ms data for  all  ions present ( figure 1 ) . in this manner , ms greatly expands the number of peptides detected using limited lc - separation compared to dda on qtof type instruments ( geromanos et al . , 2009 ) .
a drawback of ms is its incompatibility with quantitation schemes that make use of an amine reactive isotopic - label and specific reporter fragment ions to ascertain protein quantity such as itraq ( isobaric tag for relative and absolute quantification ; wiese et al . , 2007 ) .
this approach was used very recently for quantitation of early effects of hiv infection ( navare et al . ,
2012 ) using multi - dimensional separation and an orbitrap mass spectrometer ( makarov and scigelova , 2010 ) in which 1448 proteins were reliably quantified .
ms is well suited for label - free quantitation and pilot studies applying it to our model system ( uninfected pm1 t cells vs. cells at the peak of hiv-1 infection ) are promising .
so far we could quantify 358 proteins , with at least 16 proteins clearly up- or downregulated ( more than twofold ) .
consistent with our previous observations these were found either to be hardly changed or downregulated .
several other proteins found to be changed in abundance previously ( ringrose et al . , 2008 ) were again detected , but whereas , e.g. stathmin ( q96ce4 ) is downregulated as before , several 14 - 3 - 3 proteins are now upregulated instead of downregulated ( see discussion ) .
total numbers of identified proteins are obviously lower than in the 2d - dige approach , but the technique is much faster , and less labor intensive ( days vs. months ) .
also , as lower amounts of protein are needed for analysis , smaller and more reproducible cell culture samples can be used . in the future we plan to combine this approach with in - line enrichment of phosphopeptides using titanium dioxide chromatography ( pinkse et al . , 2004 , 2011 ) to look at changes in the cellular phosphoproteome upon hiv-1 infection .
in addition , lc  ms will be used with cell lines containing an inducible hiv-1 provirus ( jeeninga et al . , 2008 ) .
this allows a more synchronous induction of virus production compared to viral infection , increasing the sensitivity of the assay such that small biological changes can be detected . this will also make it feasible to discriminate between changes induced by the initial virus infection and the subsequent stage of new virus production
follow - up study on some of the proteins identified in the 2d - dige study was performed with an rna interference ( rnai ) knockdown screen .
protein induction may reflect host defensive mechanisms to prevent or restrict virus infection or replication .
alternatively , such changes may represent a viral strategy to induce cellular factors facilitating specific steps of the replication cycle ( cofactors ) .
for 76 cellular targets the impact on hiv-1 replication was studied upon mrna knockdown , using short hairpin rna ( shrna ) inhibitors from the mission library ( moffat et al . , 2006 ) .
for each target gene four to five shrnas to generate stably transduced t cells were used , thus reducing the chance of scoring off - target effects .
knockdown of 38 individual mrna targets resulted in decreased virus replication , possibly because of suppression of a viral cofactor .
of these , 27 proteins were upregulated during hiv-1 infection in our previous 2d - dige proteomic screen , fitting the cofactor role . for three targets
an increase in viral replication was observed , raising the possibility that a viral restriction factor was hit ( unpublished results ) .
as mentioned above , one of the most severe limitations of the 2d - dige pmf approach lies in the fact that about two - thirds of all the differentially expressed proteins detected can not be identified using pmf , as they are not sufficiently abundant .
this reflects the major challenge in all proteomic studies : identification and ( relative ) quantification of proteins with lower abundancies .
we detected 1920 spots , of which 15% ( 288 ) were differentially expressed at 710 days post - infection ( p.i . ;
however , we have some additional information regarding these unidentified protein spots : we know the pi and mw of the protein , i.e. of the specific isoform(s ) detected , which in most cases represent the most abundant , mature protein form(s ) .
we can also surmise what pathways the proteins most likely are involved in , based on the results obtained for the ~100 identified spots . using this information we are developing bioinformatic algorithms to come up with accurate lists of candidate differentially expressed proteins upon virus infection .
obviously , such candidates have to be confirmed experimentally , checked for instance with highly sensitive antibody - based methods such as western blotting .
first , for the pmf - identified proteins the theoretical pi and mw are computed for the mature form using  compute pi / mw  .
second , two non - linear models are fitted to predict pi and mw , respectively
. parameters of the model are estimated from the x / y - coordinates of the identified spots and the pi and mw determined in the previous step .
third , for each unidentified spot a list of candidate proteins is determined using the estimated pi and mw as input for tagident .
because of uncertainty in the pi and mw values , we choose relatively large windows so that the candidate list is likely to include the correct protein .
this is addressed in the last two steps , using the principle of  guilt by association  : more likely candidate proteins share more features with already identified proteins , e.g. , being present in the same pathway . in the fourth step of our algorithm
the physical and/or functional interactions of the ~100 identified proteins are extracted from the search tool for the retrieval of interacting genes ( string ) database ( szklarczyk et al . , 2011 ) .
protein interactions , co - occurrence in the abstracts of scientific reports , etc . , and provides confidence scores for strengths of the associations . in step 5 , candidate proteins are ranked using confidence scores by summing weighted interactions with our  identified protein  string set . by combining 2d - dige with this kind of bioinformatic algorithm
it would become one of the first techniques able to identify differential proteins in the lower regions of the dynamic range .   how to identify candidate proteins based on pi and mw .
t cell interaction , it is observed that the various approaches yield a wide range in reported numbers of quantifiable proteins , ranging from 3255 ( chan et al . , 2007 ) and 1448 ( navare et al . ,
2012 ) in total quantifiable proteins for techniques using multi - dimensional separation of peptides to 92 differentially expressed proteins ( out of 1,920 spots ) in a 2d - dige approach ( ringrose et al . , 2008 ) .
in contrast , the numbers of differentially expressed proteins are somewhat comparable at various times p.i .
( 2008 ) reported 9 ( 42 h p.i . ) and 92 ( 710 days p.i . )
regulated proteins upon infection , while the group of katze detected 687 ( 36 h p.i . ) changed proteins ( chan et al . , 2007 ) and found 266 ( 4 h p.i . ) , 60 ( 8 h p.i . ) , and 22 ( 20 h p.i . ) proteins differentially expressed earlier on in infection ( navare et al . , 2012 ) .
although numbers can of course vary according to statistical significance settings , only a small subset of proteins is usually identified by all methods ( fahey et al . , 2011 ) .
each experimental setup yields a considerable group of proteins that are not scored by the other methods .
this complementarity can be explained not only by differences in detection and quantification methods , but frequently by differences in the experimental biological systems as well .
variations include : host cell type and virus isolate ( e.g. , with a different receptor use and cell tropism),and timepoint of sampling .
it can even determine whether differentially expressed protein(form)s are found to be up- or downregulated in the interaction between hiv-1 and its host .
14 - 3 - 3 protein epsilon ( p62258 ) is a case in point : it is upregulated after 4 h p.i . and downregulated later on at 710 days p.i .
three other 14 - 3 - 3 proteins now seem to follow suit : tau / theta ( p27348 ) , gamma ( p61981 ) , and zeta / delta ( p63104 ) are found to be upregulated in our latest ms experiments , but were clearly found to be downregulated in ( ringrose et al . , 2008 ) .
these proteins can be phosphorylated on serine as well as threonine , e.g. , influencing their migration on 2d , and consequently pattern and abundance changes can be difficult to interpret .
as mentioned , an interesting recent study to look at the earliest events in hiv infection characterizing the host response at the protein level in cd4 sup - t1 cells 4 , 8 , and 20 h p.i . using hiv-1
just a few examples : high - mobility group box 1 ( p09429 ) and cofilin ( p23528 ) are strongly upregulated 4 h p.i .
, return to  normal  4 and 16 h later , while at 710 days p.i .
both are downregulated ; glucose-6-phosphate isomerase ( p06744 ) is upregulated at 8 h p.i . , but at 710 days p.i .
is found to be downregulated . given all this complexity , it is safe to say that proteomic studies on hiv-1 in general , and on hiv  t cell interaction in particular ,
but it will not be easy to translate these snapshot datasets into a comprehensive mechanistic understanding of all interactions involved ( haarburger and pillay , 2010 ; zhang et al . , 2010 ) .
as mentioned , another problem of proteomic studies is identification and quantification of proteins with lower abundancies .
one of the possible solutions to do this in a relatively unbiased fashion is sampling a proteome via interaction with random hexameric peptides using arg , lys , his , phe , tyr , trp , leu , and val only : proteominer beads ( boschetti and righetti , 2008 ) .
samples still seem to be dominated by the most abundant proteins , however .  looking at less to see more  might be the better way ahead
. such focusing could be on the analysis of specific cellular fractions ( e.g. mitochondrial preparations ) , or on the selective study of specific classes of protein or ptms , such as the phosphoproteome mentioned above .
another example of zooming in on specific protein subsets is the use of methods to enrich for cellular factors that directly interact with hiv-1 proteins .
the most general approach was performed with tagged versions of all 18 hiv-1 ( poly)proteins .
the accessory factors vif , vpu , vpr , and nef , tat and rev , as well as the polyproteins gag , pol , and gp160 , and their processed products ( ma , ca , nc , and p6 ; pr , rt , and in ; gp120 and gp41 , respectively ) were used as bait .
vif interactomics revealed how vif targets the antiviral apobec3 g protein for degradation via cbf- , using the method just described ( jager et al .
the rev protein was used as bait to fish for partners in hela cell extracts , which were then analyzed by mudpit ( multidimensional protein identification technology ) lc  ms / ms ( naji et al . , 2012 ) .
 indirect  interactomics has also been performed by expressing either wild - type vpu or vpu that is unable to associate with f - box protein -trcp in hela cells . without this interaction vpu
potential targets of vpu were then identified by quantitative proteomics using silac ( stable isotope labeling by amino acids in cell culture ) followed by lc 
much attention has been focused on the multiple roles of the ca ( capsid ) protein ( mascarenhas and musier - forsyth , 2009 ) and the tat protein ( sobhian et al .
global approaches to identify host cofactors usually consist of screening for reduced viral replication upon rnai knockdown , or enhanced replication in case a cellular restriction factor is hit ( zhou et al .
such genome wide rnai screens can easily lead to both false positives ( by off - target effects ) and false negatives ( by inefficient knockdown ) .
these considerations emphasize the importance of performing a concerted multi - disciplinary experimental approach , including gene expression ( transcriptome analysis ) , rnai and proteomic studies using different detection and labeling techniques . at the same time
, such a wider survey will generate more and larger datasets in formats which are not at all easily compared : an enormous future challenge for bioinformaticians . in light of this
we also stress the importance of specific , non  omic ,  hypothesis driven follow - up research : not generating large new datasets but asking highly specific questions .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
 this mini - review summarizes techniques applied in , and results obtained with , proteomic studies of human immunodeficiency virus type 1 ( hiv-1)t cell interaction . 
 our group previously reported on the use of two - dimensional differential gel electrophoresis ( 2d - dige ) coupled to matrix assisted laser - desorption time of flight peptide mass fingerprint analysis , to study t cell responses upon hiv-1 infection . 
 only one in three differentially expressed proteins could be identified using this experimental setup . here 
 we report on our latest efforts to test models generated by this data set and extend its analysis by using novel bioinformatic algorithms . 
 the 2d - dige results are compared with other studies including a pilot study using one - dimensional peptide separation coupled to mse , a novel mass spectrometric approach . 
 it can be concluded that although the latter method detects fewer proteins , it is much faster and less labor intensive . 
 last but not least , recent developments and remaining challenges in the field of proteomic studies of hiv-1 infection and proteomics in general are discussed . 
INTRODUCTION
A PILOT STUDY USING LCMS
FOLLOW-UP RESEARCH USING RNAi-MEDIATED KNOCKDOWN OF CELL FACTORS
BIOINFORMATIC ANALYSIS OF 2D-DIGE DATA
DISCUSSION
Conflict of Interest Statement