Targeted quantitative phosphoproteomic analysis of erythrocyte membranes during blood bank storage

One of the hallmarks of blood bank stored red blood cells (RBCs) is the irreversible transition from a discoid to a spherocyte‐like morphology with membrane perturbation and cytoskeleton disorders. Therefore, identification of the storage‐associated modifications in the protein–protein interactions between the cytoskeleton and the lipid bilayer may contribute to enlighten the molecular mechanisms involved in the alterations of mechanical properties of stored RBCs. Here we report the results obtained analyzing RBCs after 0, 21 and 35 days of storage under standard blood banking conditions by label free mass spectrometry (MS)‐based experiments. We could quantitatively measure changes in the phosphorylation level of crucial phosphopeptides belonging to β‐spectrin, ankyrin‐1, α‐adducin, dematin, glycophorin A and glycophorin C proteins. Data have been validated by both western blotting and pseudo‐Multiple Reaction Monitoring (MRM). Although each phosphopeptide showed a distinctive trend, a sharp increase in the phosphorylation level during the storage duration was observed. Phosphopeptide mapping and structural modeling analysis indicated that the phosphorylated residues localize in protein functional domains fundamental for the maintenance of membrane structural integrity. Along with previous morphological evidence acquired by electron microscopy, our results seem to indicate that 21‐day storage may represent a key point for the molecular processes leading to the erythrocyte deformability reduction observed during blood storage. These findings could therefore be helpful in understanding and preventing the morphology‐linked mechanisms responsible for the post‐transfusion survival of preserved RBCs. Copyright © 2015 John Wiley & Sons, Ltd.     

Resolution and identification of the protein components of the photosystem II antenna system of higher plants by reversed-phase liquid chromatography with electrospray-mass spectrometric detection

Reversed-phase liquid chromatography (RPLC) was interfaced to mass spectrometry (MS) with an electrospray ion (ESI) source for the separation and accurate molecular mass determination of the individual intrinsic membrane proteins that comprise the photosystem II (PS II) major light-harvesting complex (LHC II) and minor (CP24, CP26 and CP29) antenna system, whose molecular masses range between 22,000 and 29,000. PS II is a supramolecular complex intrinsic of the thylacoid membrane, which plays the important role in photosynthesis of capturing solar energy, and transferring it to photochemical reaction centers where energy conversion occurs. The protein components of the PS II major and minor antenna systems were extracted from spinach thylacoid membranes and separated using a butyl-silica column eluted by an acetonitrile gradient in 0.05% (v/v) aqueous trifluoroacetic acid. On-line electrospray MS allowed accurate molecular mass determination and identification of the protein components of PS II major and minor antenna system. The proposed RPLC-ESI-MS method holds several advantages over sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the conventional technique for studying membrane proteins, including a better protein separation, mass accuracy, speed and efficiency.     

Intact mass measurements for unequivocal identification of hydrophobic photosynthetic photosystems I and II antenna proteins

The purpose of this study was to determine the optimal experimental conditions necessary to allow rapid and accurate identification of highly hydrophobic proteins, such as the antenna proteins from photosystems I and II. The antenna proteins were derived from two different species, tomato and Arabidopsis, whose photosynthetic genome is well known. The separation techniques included sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting, microamino acid sequencing, reversed-phase high-performance liquid chromatography (RP-HPLC), intact mass measurements, and peptide mass fingerprinting by mass spectrometry. Immunoblotting was time-consuming, and success was limited due to cross-reactivity between these highly conserved sequences. The best results by far were achieved by separating intact proteins through hyphenation of reversed-phase liquid chromatography on-line to an electrospray ionization mass spectrometer (ESI-MS) and consequently identifying individual proteins from their intact mass measurements (IMMs), whereas peptide mass fingerprinting was hampered by the highly hydrophobic nature of these proteins. RP-HPLC-ESI-MS was superior in the quality of separation. Moreover, the high quality of mass spectra recorded during the RP-HPLC-ESI-MS analysis meant that the relative deviations of the molecular masses determined with a quadrupole ion trap mass analyzer ranged between 100 and 300 ppm. Thus, the correspondence between the intact mass values measured with those deduced from the DNA sequences allowed the different types of antenna proteins to be identified and assigned to their corresponding gene families. By utilizing this correlation, it was possible to spot gene products of previously cloned genes.     

Separation and identification of photosynthetic antenna membrane proteins by high- performance liquid chromatography electrospray ionization mass spectrometry

Functional proteomics of membrane proteins is an important tool for the understanding of protein networks in biological membranes. Nevertheless, structural studies on this part of the proteome are limited. The present review attempts to cover the vast array of methods that have appeared in the last few years for separation and identification of photosynthetic proteins of thylakoid membranes present in chloroplasts, a good model for setting up analytical methods suitable for membrane proteins. The two major methods for the separation of thylakoid membrane proteins are gel electrophoresis and liquid chromatography. Isoelectric focusing in a first dimension followed by denaturing sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) in a second dimension is an effective way to resolve large numbers of soluble and peripheral membrane proteins. However, it is not applicable for isolation of native protein complexes or for the separation of highly hydrophobic membrane proteins. High-performance liquid chromatography (HPLC), on the other hand, is highly suitable for any type of membrane protein separation due to its compatibility with detergents that are necessary to keep the hydrophobic proteins in solution. With regard to the identification of the separated proteins, several methods are available, including immunological and mass spectrometric methods. Besides immunological identification, peptide mass fingerprinting, peptide fragment fingerprinting or intact molecular mass determination by electrospray ionization mass spectrometry (ESI-MS) or matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) have been shown to be very sensitive and effective. In particular, identification of proteins by their intact molecular mass is advantageous for the investigation of numerous biological problems, because it is rapid and reflects the full sequence of the protein and all its posttranslational modifications. However, intact molecular mass determinations of gel-separated membrane proteins are hampered due to the difficulties in extracting the hydrophobic proteins from the gel, whereas HPLC on-line interfaced with ESI-MS enables the rapid and accurate determination of intact molecular masses and consequently an unequivocal protein identification. This strategy can be viewed as a multidimensional separation technique distinguishing between hydrophobicity in the first dimension and between different mass-to-charge ratios in the second dimension, allowing the separation and identification even of isomeric forms.     

Separation and identification of the light harvesting proteins contained in grana and stroma thylakoid membrane fractions

This paper presents the results of a study performed to develop a rapid and straightforward method to resolve and simultaneously identify the light-harvesting proteins of photosystem I (LHCI) and photosystem II (LHCII) present in the grana and stroma of the thylakoid membranes of higher plants. These hydrophobic proteins are embedded in the phospholipid membrane, and their extraction usually requires detergent and time consuming manipulations that may introduce artifacts. The method presented here makes use of digitonin, a detergent which causes rapid (within less than 3 min) cleavage of the thylakoid membrane into two subfractions: appressed (grana) and non-appressed (stroma) membranes, the former enriched in photosystem II and the latter containing mainly photosystem I. From these two fractions identification of the protein components was performed by separating them by reversed-phase high-performance liquid chromatography (RP-HPLC) and determining the intact molecular mass by electrospray ionization mass spectrometry (ESI-MS). By this strategy the ion suppression during ESI-MS that normally occurs in the presence of membrane phospholipids was avoided, since RP-HPLC removed most phospholipids from the analytes. Consequently, high quality mass spectra were extracted from the reconstructed ion chromatograms. The specific cleavage of thylakoid membranes by digitonin, as well as the rapid identification and quantification of the antenna composition of the two complexes facilitate future studies of the lateral migration of the chlorophyll-protein complexes along thylakoid membranes, which is well known to be induced by high intensity light or other environmental stresses. Such investigations could not be performed by sodium dodecylsulfate-polyacrylamide gel electrophoresis because of insufficient resolution of the proteins having molecular masses between 22,000 and 25,000.     

De novo sequence analysis and intact mass measurements for characterization of phycocyanin subunit isoforms from the blue‐green alga Aphanizomenon flos‐aquae

In this work, partial characterization of the primary structure of phycocyanin from the cyanobacterium Aphanizomenon flos‐aquae (AFA) was achieved by mass spectrometry de novo sequencing with the aid of chemical derivatization. Combining N‐terminal sulfonation of tryptic peptides by 4‐sulfophenyl isothiocyanate (SPITC) and MALDI‐TOF/TOF analyses, facilitated the acquisition of sequence information for AFA phycocyanin subunits. In fact, SPITC‐derivatized peptides underwent facile fragmentation, predominantly resulting in y‐series ions in the MS/MS spectra and often exhibiting uninterrupted sequences of 20 or more amino acid residues. This strategy allowed us to carry out peptide fragment fingerprinting and de novo sequencing of several peptides belonging to both α‐ and β‐phycocyanin polypeptides, obtaining a sequence coverage of 67% and 75%, respectively. The presence of different isoforms of phycocyanin subunits was also revealed; subsequently Intact Mass Measurements (IMMs) by both MALDI‐ and ESI‐MS supported the detection of these protein isoforms. Finally, we discuss the evolutionary importance of phycocyanin isoforms in cyanobacteria, suggesting the possible use of the phycocyanin operon for a correct taxonomic identity of this species. Copyright © 2008 John Wiley & Sons, Ltd.     

Proteomics for quality-control processes in transfusion medicine

Following the publication of recent retrospective and highly debated studies, transfusionists are increasingly asking for improved tools to assess the quality of transfusion-relevant products. At its dawn, proteomics emerged as a potential candidate for in-depth investigations of blood components and plasma derivatives. As its maturity is now at hand, the proteomic expertise seems to be ready to be massively transferred to the clinical setting, where it could be potentially used as a valid tool to test, from bench to bedside, the quality of collected blood components prior to or during storage. Proteomic strategies have been demonstrated to be also suited to verify the effects of the production and pathogen-reduction processes of plasma derivatives and blood components on the protein fractions, or to discover particular biomarkers readily adoptable for targeted evaluation of blood-component integrity or functionality. Although the technical background is in continuous and rapid expansion, the spread of proteomics in clinical routine practice has been hitherto hampered by high costs for dedicated facilities and specialized personnel.     

An easy preparative gel electrophoretic method for targeted depletion of hemoglobin in erythrocyte cytosolic samples

Hemoglobin (Hb) approximately constitutes 98% of the protein composition of a red blood cell (RBC), thus masking the remaining 2% which has still to be discovered completely due to the difficulty in its analysis. Here, we proposed a large-scale native gel electrophoresis that effectively tackles this limitation through a novel sample preparation strategy able to concentrate low-abundance species by removing Hb by means of electrophoretic instruments. Clear native PAGE was performed in a gel electrophoresis tube where the run was intermittently interrupted and different fractions were recovered in liquid phase into a collection chamber placed at the end of the tube. In this way, fractions containing multi-protein complexes with different molecular weights were collected in the native form by a simple elution. Red fraction containing Hb multi-protein complexes can be excluded from subsequent analyses, or rather be analyzed separately, reducing therefore the dynamic range of erythrocyte cytosolic protein concentrations and increasing the number of protein identifications. In particular, 838 protein spots in total were detected when fractions were analyzed by 2-D IEF-SDS-PAGE. This depletion method is inexpensive, simple to perform, reproducible and makes it possible to process large amounts of sample (up to 150 mg), thus making it suitable for in-depth proteome investigations. Furthermore, this strategy has the potential to be applied both to native and denatured proteomes of different biological samples.     

Plasma-derived clotting factor VIII: heterogeneity evaluation in the quest for potential inhibitory-antibody stimulating factors

In this study, we investigated the heterogeneity and the purity grade of three commercially available plasma‐derived clotting factor VIII (FVIII) concentrates, which highly differ with regard to purification strategies, relative concentrations of stabilizers (von Willebrand factor, with or without albumin) and virus inactivation strategies (solvent/detergent and/or heat/pasteurization treatments). Western blot analyses were used to evaluate product‐specific variations from Emoclot^®, Alphanate^® and Haemate^® both in the presence and absence of reducing agents (dithiotreithol). All the plasma‐derived concentrates showed a strong heterogeneity, as they all included a significant amount of truncated forms of the full‐length (FL) clotting FVIII protein. The intact protein accounted for the 38% of the total FVIII proteins in Haemate^® and 29 and 23% in Alphanate^® and Emoclot^®, respectively. Lower intact FVIII amounts in Emoclot might be mainly due to the low von Willebrand factor dosage and the absence of albumin. Upon addition of thrombin, both the FL and truncated forms of the FVIII protein were almost completely digested. Indeed, after thrombin activation, we could still observe a mixture of B‐domain truncated forms of the FL protein along with biologically active digested‐A1 forms. Batch‐to‐batch variation was tested with no evident changes appearing among different batches. Despite the variables in manufacturing processes, inter‐product comparisons yielded similar results for all the plasma‐derived FVIII considered in this study. However, we could individuate in Emoclot a band that was not digested by thrombin, which we could characterize as the 200 kDa FVIII heavy chain. This investigation prompts new concerns about the strong heterogeneity observed upon thrombin digestion of plasma‐derived FVIII, which might contribute to the development of inhibitory antibodies at an early stage of therapy, and to which extent these untoward phenomena could be avoided through direct intervention on routine manufacturing processes.